Interaction between poly(A)-binding protein PABPC4 and nuclear receptor corepressor NCoR1 modulates a metabolic stress response.

Mitochondria are organelles known primarily for generating ATP via the oxidative phosphorylation process. Environmental signals are sensed by whole organisms or cells and markedly affect this process, leading to alterations in gene transcription and, consequently, changes in mitochondrial function and biogenesis. The expression of mitochondrial genes is finely regulated by nuclear transcription factors, including nuclear receptors and their coregulators. Among the best-known coregulators is the nuclear receptor corepressor 1 (NCoR1). Muscle-specific knockout of NCoR1 in mice induces an oxidative phenotype, improving glucose and fatty acid metabolism. However, the mechanism by which NCoR1 is regulated remains elusive. In this work, we identified the poly(A)-binding protein 4 (PABPC4) as a new NCoR1 interactor. Unexpectedly, we found that silencing of PABPC4 induced an oxidative phenotype in both C2C12 and MEF cells, as indicated by increased oxygen consumption, mitochondria content, and reduced lactate production. Mechanistically, we demonstrated that PABPC4 silencing increased the ubiquitination and consequent degradation of NCoR1, leading to the derepression of PPAR-regulated genes. As a consequence, cells with PABPC4 silencing had a greater capacity to metabolize lipids, reduced intracellular lipid droplets, and reduced cell death. Interestingly, in conditions known to induce mitochondrial function and biogenesis, both mRNA expression and PABPC4 protein content were markedly reduced. Our study, therefore, suggests that the lowering of PABPC4 expression may represent an adaptive event required to induce mitochondrial activity in response to metabolic stress in skeletal muscle cells. As such, the NCoR1-PABPC4 interface might be a new road to the treatment of metabolic diseases.

Estrogen-related receptor gamma regulates mitochondrial and synaptic genes and modulates vulnerability to synucleinopathy.

Many studies implicate mitochondrial dysfunction as a key contributor to cell loss in Parkinson disease (PD). Previous analyses of dopaminergic (DAergic) neurons from patients with Lewy-body pathology revealed a deficiency in nuclear-encoded genes for mitochondrial respiration, many of which are targets for the transcription factor estrogen-related receptor gamma (Esrrg/ERRγ). We demonstrate that deletion of ERRγ from DAergic neurons in adult mice was sufficient to cause a levodopa-responsive PD-like phenotype with reductions in mitochondrial gene expression and number, that partial deficiency of ERRγ hastens synuclein-mediated toxicity, and that ERRγ overexpression reduces inclusion load and delays synuclein-mediated cell loss. While ERRγ deletion did not fully recapitulate the transcriptional alterations observed in postmortem tissue, it caused reductions in genes involved in synaptic and mitochondrial function and autophagy. Altogether, these experiments suggest that ERRγ-deficient mice could provide a model for understanding the regulation of transcription in DAergic neurons and that amplifying ERRγ-mediated transcriptional programs should be considered as a strategy to promote DAergic maintenance in PD.

The Detection of Early Epigenetic Inheritance of Mitochondrial Stress in C. Elegans with a Microfluidic Phenotyping Platform.

Fluctuations and deterioration in environmental conditions potentially have a phenotypic impact that extends over generations. Transgenerational epigenetics is the defined term for such intergenerational transient inheritance without an alteration in the DNA sequence. The model organism Caenorhabditis elegans is exceptionally valuable to address transgenerational epigenetics due to its short lifespan, well-mapped genome and hermaphrodite behavior. While the majority of the transgenerational epigenetics on the nematodes focuses on generations-wide heritage, short-term and in-depth analysis of this phenomenon in a well-controlled manner has been lacking. Here, we present a novel microfluidic platform to observe mother-to-progeny heritable transmission in C. elegans at high imaging resolution, under significant automation, and enabling parallelized studies. After approximately 24 hours of culture of L4 larvae under various concentrations and application periods of doxycycline, we investigated if mitochondrial stress was transferred from the mother nematodes to the early progenies. Automated and custom phenotyping algorithms revealed that a minimum doxycycline concentration of 30 µg/mL and a drug exposure time of 15 hours applied to the mothers could induce mitochondrial stress in first embryo progenies indeed, while this inheritance was not clearly observed later in L1 progenies. We believe that our new device could find further usage in transgenerational epigenetic studies modeled on C. elegans.

Automated Platform for Long-Term Culture and High-Content Phenotyping of Single C. elegans Worms.

The nematode Caenorhabditis elegans is a suitable model organism in drug screening. Traditionally worms are grown on agar plates, posing many challenges for long-term culture and phenotyping of animals under identical conditions. Microfluidics allows for 'personalized' phenotyping, as microfluidic chips permit collecting individual responses over worms' full life. Here, we present a multiplexed, high-throughput, high-resolution microfluidic approach to culture C. elegans from embryo to the adult stage at single animal resolution. We allocated single embryos to growth chambers, for observing the main embryonic and post-embryonic development stages and phenotypes, while exposing worms to up to 8 different well-controlled chemical conditions. Our approach allowed eliminating bacteria aggregation and biofilm formation-related clogging issues, which enabled us performing up to 80 hours of automated single worm culture studies. Our microfluidic platform is linked with an automated phenotyping code that registers organism-associated phenotypes at high-throughput. We validated our platform with a dose-response study of the anthelmintic drug tetramisole by studying its influence through the life cycle of the nematodes. In parallel, we could observe development effects and variations in single embryo and worm viability due to the bleaching procedure that is standardly used for harvesting the embryos from a worm culture agar plate.

Miniaturized implantable sensors for in vivo localized temperature measurements in mice during cold exposure.

We report on in vivo temperature measurements performed in mice at two specific sites of interest in the animal body over a period of several hours. In particular, the aim of this work was to monitor mouse metabolism during cold exposure, and to record possible temperature differences between the body temperature measured in the abdomen and the temperature of the brown adipose tissue (BAT) situated in the interscapular area. This approach is of biological interest as it may help unravelling the question whether biochemical activation of BAT is associated with local increase in metabolic heat production. For that purpose, miniaturized thermistor sensors have been accurately calibrated and implanted in the BAT and in the abdominal tissue of mice. After 1 week of recovery from surgery, mice were exposed to cold (6 °C) for a maximum duration of 6 h and the temperature was acquired continuously from the two sensors. Control measurements with a conventional rectal probe confirmed good performance of both sensors. Moreover, two different mouse phenotypes could be identified, distinguishable in terms of their metabolic resistance to cold exposure. This difference was analyzed from the thermal point of view by computational simulations. Our simple physical model of the mouse body allowed to reproduce the global evolution of hypothermia and also to explain qualitatively the temperature difference between abdomen and BAT locations. While with our approach, we have demonstrated the importance and feasibility of localized temperature measurements on mice, further optimization of this technique may help better identify local metabolism variations.

Conjugated bile acids associate with altered rates of glucose and lipid oxidation after Roux-en-Y gastric bypass.

BACKGROUND: Laparoscopic Roux-en-Y gastric bypass (RYGB) induces a more favorable metabolic profile than expected by weight loss alone. In this study, we investigated the effect of RYGB on serum bile acid levels and their relation to clinical outcomes. METHODS: We included 30 obese patients who underwent RYGB (BMI = 46.1 ± 5.9 kg/m(2)). Clinical measurements and laboratory determinations were performed before surgery and 1 year after surgery. Fasting serum bile acids were measured by an enzymatic method and individual bile acids were quantified by HLPC-tandem mass spectrometry. Indirect calorimetry was performed to measure the rates of energy expenditure and substrate oxidation. RESULTS: Fasting total serum bile acid levels increased twofold after RYGB (pre, 3.68 ± 2.03 vs. post, 7.06 ± 9.65 µmol/l, +92 %, p = 0.002). This increase in total bile acids was accompanied by a decrease in conjugated bile acids, which correlated with decreased glucose oxidation (r = 0.571, p = 0.002) and with increased lipid oxidation (r = -0.626, p = 0.0004). The change in taurine-conjugated bile acids correlated with altered DIO2 mRNA expression in adipose tissue (r = -0.498, p = 0.013) potentially linking bile acid conjugation to substrate oxidation through DIO2. CONCLUSIONS: Fasting serum bile acid levels increase after RYGB. More specifically, changes in bile acid conjugation after RYGB associate with altered energy metabolism.

NAD+ as a signaling molecule modulating metabolism.

The ability of NAD(+) to act as a metabolic cofactor and as a rate-limiting cosubstrate for many enzymes, particularly the sirtuins, has led to the identification of a pivotal role of NAD(+) levels in the control of whole-body metabolic homeostasis. Bioavailability and compartmentalization of NAD(+) have become highly relevant issues that we need to understand in order to elucidate how NAD(+) acts both as a readout of the metabolic milieu and as an effector triggering appropriate cellular adaptations.

Reversible acetylation of PGC-1: connecting energy sensors and effectors to guarantee metabolic flexibility.

Organisms adapt their metabolism to meet ever changing environmental conditions. This metabolic adaptation involves at a cellular level the fine tuning of mitochondrial function, which is mainly under the control of the transcriptional co-activator proliferator-activated receptor gamma co-activator (PGC)-1alpha. Changes in PGC-1alpha activity coordinate a transcriptional response, which boosts mitochondrial activity in times of energy needs and attenuates it when energy demands are low. Reversible acetylation has emerged as a key way to alter PGC-1alpha activity. Although it is well established that PGC-1alpha is deacetylated and activated by Sirt1 and acetylated and inhibited by GCN5, less is known regarding how these enzymes themselves are regulated. Recently, it became clear that the energy sensor, AMP-activated kinase (AMPK) translates the effects of energy stress into altered Sirt1 activity by regulating the intracellular level of its co-substrate nicotinamide adenine dinucleotide (NAD)(+). Conversely, the enzyme ATP citrate lyase (ACL), relates energy balance to GCN5, through the control of the nuclear production of acetyl-CoA, the substrate for GCN5's acetyltransferase activity. We review here how these metabolic signaling pathways, affecting GCN5 and Sirt1 activity, allow the reversible acetylation-deacetylation of PGC-1alpha and the adaptation of mitochondrial energy homeostasis to energy levels.

Targeting the TGR5-GLP-1 pathway to combat type 2 diabetes and non-alcoholic fatty liver disease.

Incretin-based therapies have shown promise in the treatment of type 2 diabetes. Here we review our current understanding of TGR5 as a target to induce glucagon-like peptide-1 (GLP-1) secretion. These new observations suggest that TGR5 agonists may constitute a novel approach to treat type 2 diabetes, as well as complications of diabetes, such as non-alcoholic fatty liver disease.

Scaffold attachment factor B1 directly interacts with nuclear receptors in living cells and represses transcriptional activity.

Transcriptional activity relies on coregulators that modify the chromatin structure and serve as bridging factors between transcription factors and the basal transcription machinery. Using the DE domain of human peroxisome proliferator-activated receptor gamma (PPARgamma) as bait in a yeast two-hybrid screen of a human adipose tissue library, we isolated the scaffold attachment factor B1 (SAFB1/HET/HAP), which was previously shown to be a corepressor of estrogen receptor alpha. We show here that SAFB1 has a very broad tissue expression profile in human and is also expressed all along mouse embryogenesis. SAFB1 interacts in pull-down assays not only with PPARgamma but also with all nuclear receptors tested so far, albeit with different affinities. The association of SAFB1 and PPARgamma in vivo is further demonstrated by fluorescence resonance energy transfer (FRET) experiments in living cells. We finally show that SAFB1 is a rather general corepressor for nuclear receptors. Its change in expression during the early phases of adipocyte and enterocyte differentiation suggests that SAFB1 potentially influences cell proliferation and differentiation decisions.

Peroxisome proliferator-activated receptor gamma: the more the merrier?

The consequence of activating the nuclear hormone receptor, peroxisome proliferator-activated receptor gamma (PPARgamma), which coordinates adipocyte differentiation, validates the concept, 'you are what you eat'. Excessive caloric intake leads to fat formation if the energy from these nutrients is not expended. However, this evolutionary adaptation to store energy in fat, which can be released under the form of fatty acids, potent PPARgamma agonists, has become a disadvantage in today's affluent society as it results in numerous metabolic imbalances, collectively known as the metabolic syndrome. With the surge of human and genetic studies on PPARgamma function, the limitations to the benefits of PPARgamma signalling have been realized. It is now evident that the most effective strategy for resetting the balance of this thrifty gene is through its modulation rather than full activation, with the goal to improve glucose homeostasis while preventing adipogenesis. Finally, as more PPARgamma targeted pathways are revealed such as bone homeostasis, atherosclerosis and longevity, it is most certain that the PPARgamma thrifty gene hypothesis will evolve to incorporate these.

Adipose tissue: new therapeutic targets from molecular and genetic studies--IASO Stock Conference 2003 report.

This review highlights the presentations and discussions held during the 2003 Stock Conference in Lisbon focussed on the identification of new therapeutic targets for the treatment of obesity and identified through molecular and genetic studies. Transcription factors and their cofactors, signalling pathways and new insights provided by cellular and genetic studies were discussed as potential new avenues to modulate adipocyte formation and function.

Regulation of adipocyte differentiation.

Once multipotent mesenchymal cells become committed to the adipoblast lineage, adipogenesis, the process of preadipocytes differentiation into adipocytes is initiated. This process starts with a phase of exponential growth of adipoblasts. Following confluence of these adipoblasts, the cells enter into a cell cycle arrest, they re-enter the cell cycle and pass through a limited number of cell divisions, and finally differentiate into fully mature adipocytes. Adipogenesis is controlled by a complex cross-talk between positive and negative regulators, such as hormonal and nutritional stimuli, that change the activity of a selected set of transcription factors. Regulation of adipogenesis is crucial to keep the body energy balance because a limited amount of adipose tissue, lipodystrophy, or an excess of adipose tissue, such as occurs in obesity, lead to profound metabolic dysfunctions and disease.

A unique PPARgamma ligand with potent insulin-sensitizing yet weak adipogenic activity.

FMOC-L-Leucine (F-L-Leu) is a chemically distinct PPARgamma ligand. Two molecules of F-L-Leu bind to the ligand binding domain of a single PPARgamma molecule, making its mode of receptor interaction distinct from that of other nuclear receptor ligands. F-L-Leu induces a particular allosteric configuration of PPARgamma, resulting in differential cofactor recruitment and translating in distinct pharmacological properties. F-L-Leu activates PPARgamma with a lower potency, but a similar maximal efficacy, than rosiglitazone. The particular PPARgamma configuration induced by F-L-Leu leads to a modified pattern of target gene activation. F-L-Leu improves insulin sensitivity in normal, diet-induced glucose-intolerant, and in diabetic db/db mice, yet it has a lower adipogenic activity. These biological effects suggest that F-L-Leu is a selective PPARgamma modulator that activates some (insulin sensitization), but not all (adipogenesis), PPARgamma-signaling pathways.

PPARgamma/RXRalpha heterodimers control human trophoblast invasion.

The ligand-dependent nuclear receptors PPARgamma and RXRalpha/beta were recently determined to be essential for murine placental development and trophoblast differentiation. In the current study we examined the expression and role of the PPARgamma/RXRalpha heterodimers in human invasive trophoblasts. We first report that in human first trimester placenta, PPARgamma and RXRalpha are highly expressed in cytotrophoblasts at the feto-maternal interface, especially in the extravillous cytotrophoblasts involved in uterus invasion. The coexpression of PPARgamma and RXRalpha genes in extravillous cytotrophoblast nuclei were then confirmed by immunocytochemistry, immunoblot, and real-time quantitative PCR using cultured purified primary extravillous cytotrophoblasts. We next examined, using the extravillous cytotrophoblast culture model, the biological role of PPARgamma/RXRalpha heterodimers in vitro, and we showed that both synthetic (rosiglitazone) and natural [15-deoxy-delta-(12,14)PGJ(2)] PPARgamma agonists inhibit extravillous cytotrophoblast invasion in a concentration-dependent manner and synergize with pan-RXR agonists. Conversely, PPARgamma or pan-RXR antagonists promoted extravillous cytotrophoblast invasion. Furthermore, the pan-RXR antagonist abolished the inhibitory effect of the PPARgamma agonists. Together these data underscore an important function of PPARgamma/RXRalpha heterodimers in the modulation of trophoblast invasion.

PPAR gamma/RXR alpha heterodimers are involved in human CG beta synthesis and human trophoblast differentiation.

Recent studies performed with null mice suggested a role of either RXR alpha or PPAR gamma in murine placental development. We report here that both PPAR gamma and RXR alpha are strongly expressed in human villous cytotrophoblasts and syncytiotrophoblasts. Moreover, specific ligands for RXRs or PPAR gamma (but not for PPAR alpha or PPAR delta) increase both human CG beta transcript levels and the secretion of human CG and its free beta-subunit. When combined, these ligands have an additive effect on human CG secretion. Pan-RXR and PPAR gamma ligands also have an additive effect on the synthesis of other syncytiotrophoblast hormones such as human placental lactogen, human placental GH, and leptin. Therefore, in human placenta, PPAR gamma/RXR alpha heterodimers are functional units during cytotrophoblast differentiation into the syncytiotrophoblast in vitro. Elements located in the regulatory region of the human CG beta gene (beta 5) were found to bind RXR alpha and PPAR gamma from human cytotrophoblast nuclear extracts, suggesting that PPAR gamma/RXR alpha heterodimers directly regulate human CG beta transcription. Altogether, these data show that PPAR gamma/RXR alpha heterodimers play an important role in human placental development.

Peroxisome proliferator-activated receptor-alpha regulates lipid homeostasis, but is not associated with obesity: studies with congenic mouse lines.

Considerable controversy exists in determining the role of peroxisome proliferator-activated receptor-alpha (PPARalpha) in obesity. Two purebred congenic strains of PPARalpha-null mice were developed to study the role of this receptor in modulating lipid transport and storage. Weight gain and average body weight in wild-type and PPARalpha-null mice on either an Sv/129 or a C57BL/6N background were not markedly different between genotypes from 3 to 9 months of age. However, gonadal adipose stores were significantly greater in both strains of male and female PPARalpha-null mice. Hepatic accumulation of lipids was greater in both strains and sexes of PPARalpha-null mice compared with wild-type controls. Administration of the peroxisome proliferator WY-14643 caused hepatomegaly, alterations in mRNAs encoding proteins that regulate lipid metabolism, and reduced serum triglycerides in a PPARalpha-dependent mechanism. Constitutive differences in serum cholesterol and triglycerides in PPARalpha-null mice were found between genetic backgrounds. Results from this work establish that PPARalpha is a critical modulator of lipid homeostasis in two congenic mouse lines. This study demonstrates that disruption of the murine gene encoding PPARalpha results in significant alterations in constitutive serum, hepatic, and adipose tissue lipid metabolism. However, an overt, obese phenotype in either of the two congenic strains was not observed. In contrast to earlier published work, this study establishes that PPARalpha is not associated with obesity in mice.

Peroxisome proliferator-activated receptor-gamma: from adipogenesis to carcinogenesis.

Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors, initially described as molecular targets for synthetic compounds inducing peroxisome proliferation. PPAR-gamma, the best characterized of the PPARs, plays a crucial role in adipogenesis and insulin sensitization. Furthermore, PPAR-gamma has been reported to affect cell proliferation/differentiation pathways in various malignancies. We discuss in the present review recent advances in the understanding of the function of PPAR-gamma in both cell proliferation and adipocyte differentiation.

PPAR gamma: an essential role in metabolic control.

The peroxisome proliferator-activated receptor gamma is a nuclear hormone receptor playing a crucial role in adipogenesis and insulin sensitization. Prostaglandin J2 derivatives and the antidiabetic thiazolidinediones are its respective natural and synthetic ligands. The RXR/PPAR gamma heterodimer has also been reported to have important immunomodulatory activities and its pleiotropic functions suggest wide-ranging medical implications.