CDK4 Phosphorylates AMPKα2 to Inhibit Its Activity and Repress Fatty Acid Oxidation

The roles of CDK4 in the cell cycle have been extensively studied, but less is known about the mechanisms underlying the metabolic regulation by CDK4. Here, we report that CDK4 promotes anaerobic glycolysis and represses fatty acid oxidation in mouse embryonic fibroblasts (MEFs) by targeting the AMP-activated protein kinase (AMPK). We also show that fatty acid oxidation (FAO) is specifically induced by AMPK complexes containing the α2 subunit. Moreover, we report that CDK4 represses FAO through direct phosphorylation and inhibition of AMPKα2. The expression of non-phosphorylatable AMPKα2 mutants, or the use of a CDK4 inhibitor, increased FAO rates in MEFs and myotubes. In addition, Cdk4(-/-) mice have increased oxidative metabolism and exercise capacity. Inhibition of CDK4 mimicked these alterations in normal mice, but not when skeletal muscle was AMPK deficient. This novel mechanism explains how CDK4 promotes anabolism by blocking catabolic processes (FAO) that are activated by AMPK

Rôle du récepteur nucléaire LXR et du glucagon dans la régulation du facteur de transcription ChREBP (implication dans le développement de la stéatose hépatique et de la résistance à l'insuline)

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Hepatic Gene Regulation by Glucose and Polyunsaturated Fatty Acids: A Role for ChREBP

International audienc

La régulation de l'expression des gènes par le glucose

Le glucose ne doit plus être considéré comme un simple carburant énergétique des cellules mais aussi comme une molécule de signalisation importante dans la régulation des gènes glycolytiques et lipogéniques impliqués dans la mise en réserve d'énergie sous forme de triglycérides. Les effets transcriptionnels du glucose sur les gènes des enzymes impliqués dans la glycolyse et la lipogenèse hépatiques impliquent un facteur de transcription spécifique, ChREBP, dont les caractéristiques et le mécanisme d'activation sont décrits en détail. Enfin, un exemple de l'implication possible de ChREBP dans la physiopathologie de l'obésité et du diabète de type 2 est présenté

E2F1 inhibits circulating cholesterol clearance by regulating Pcsk9 expression in the liver

International audienceCholesterol accumulation in the liver is an early event in nonalcoholic fatty liver disease (NAFLD). Here, we demonstrate that E2F1 plays a crucial role in maintaining cellular cholesterol homeostasis by regulating cholesterol uptake via proprotein convertase subtilisin/kexin 9 (PCSK9), an enzyme that promotes low-density lipoprotein receptor (LDLR) degradation upon activation. E2f1-/- mice display reduced total plasma cholesterol levels and increased cholesterol content in the liver. In this study, we show that E2f1 deletion in cellular and mouse models leads to a marked decrease in Pcsk9 expression and an increase in LDLR expression. In addition to the upregulation of LDLR, we report that E2f1-/- hepatocytes exhibit increased LDL uptake. ChIP-Seq and PCSK9 promoter reporter experiments confirmed that E2F1 binds to and transactivates the PCSK9 promoter. Interestingly, E2f1-/- mice fed a high-cholesterol diet (HCD) display a fatty liver phenotype and liver fibrosis, which is reversed by reexpression of PCSK9 in the liver. Collectively, these data indicate that E2F1 regulates cholesterol uptake and that the loss of E2F1 leads to abnormal cholesterol accumulation in the liver and the development of fibrosis in response to an HCD

E2F1 promotes hepatic gluconeogenesis and contributes to hyperglycemia during diabetes

Objective: Aberrant hepatic glucose production contributes to the development of hyperglycemia and is a hallmark of type 2 diabetes. In a recent study, we showed that the transcription factor E2F1, a component of the cell cycle machinery, contributes to hepatic steatosis through the transcriptional regulation of key lipogenic enzymes. Here, we investigate if E2F1 contributes to hyperglycemia by regulating hepatic gluconeogenesis. Methods: We use different genetic models to investigate if E2F1 regulates gluconeogenesis in primary hepatocytes and in vivo. We study the impact of depleting E2F1 or inhibiting E2F1 activity in diabetic mouse models to evaluate if this transcription factor contributes to hyperglycemia during insulin resistance. We analyze E2F1 mRNA levels in the livers of human diabetic patients to assess the relevance of E2F1 in human pathophysiology. Results: Lack of E2F1 impaired gluconeogenesis in primary hepatocytes. Conversely, E2F1 overexpression increased glucose production in hepatocytes and in mice. Several genetic models showed that the canonical CDK4-RB1-E2F1 pathway is directly involved in this regulation. E2F1 mRNA levels were increased in the livers from human diabetic patients and correlated with the expression of the gluconeogenic enzyme Pck1. Genetic invalidation or pharmacological inhibition of E2F1 improved glucose homeostasis in diabetic mouse models. Conclusions: Our study unveils that the transcription factor E2F1 contributes to mammalian glucose homeostasis by directly controlling hepatic gluconeogenesis. Together with our previous finding that E2F1 promotes hepatic steatosis, the data presented here show that E2F1 contributes to both hyperlipidemia and hyperglycemia in diabetes, suggesting that specifically targeting E2F1 in the liver could be an interesting strategy for therapies against type 2 diabetes. (C) 2018 The Authors. Published by Elsevier GmbH

Untargeted Lipidomic Profiling of Dry Blood Spots Using SFC-HRMS

International audienceLipids are essential cellular constituents that have many critical roles in physiological functions. They are notably involved in energy storage and cell signaling as second messengers, and they are major constituents of cell membranes, including lipid rafts. As a consequence, they are implicated in a large number of heterogeneous diseases, such as cancer, diabetes, neurological disorders, and inherited metabolic diseases. Due to the high structural diversity and complexity of lipid species, the presence of isomeric and isobaric lipid species, and their occurrence at a large concentration scale, a complete lipidomic profiling of biological matrices remains challenging, especially in clinical contexts. Using supercritical fluid chromatography coupled with high-resolution mass spectrometry, we have developed and validated an untargeted lipidomic approach to the profiling of plasma and blood. Moreover, we have tested the technique using the Dry Blood Spot (DBS) method and found that it allows for the easy collection of blood for analysis. To develop the method, we performed the optimization of the separation and detection of lipid species on pure standards, reference human plasma (SRM1950), whole blood, and DBS. These analyses allowed an in-house lipid data bank to be built. Using the MS-Dial software, we developed an automatic process for the relative quantification of around 500 lipids species belonging to the 6 main classes of lipids (including phospholipids, sphingolipids, free fatty acids, sterols, and fatty acyl-carnitines). Then, we compared the method using the published data for SRM 1950 and a mouse blood sample, along with another sample of the same blood collected using the DBS method. In this study, we provided a method for blood lipidomic profiling that can be used for the easy sampling of dry blood spots

Chromatin immunoprecipitation improvements for the processing of small frozen pieces of adipose tissue - Fig 1

<p>The chart shows the three different workflows performed, using the pestle and mortar (1.1), the Dounce homogenizer (1.2) or the Ultraturrax homogenizer (1.3). In (1.1), the homogenization step was performed using liquid nitrogen, after which it was fixed. After this, the nuclei were pelleted and nucleus lysis buffer was added. Once incubated, the sample was sheared and the chromatin fragmentation and recovery were checked. In the other two alternative methods proposed (1.2 and 1.3), the tissue was cut in small pieces (3 mm) and the fixation step was performed prior to the homogenization. Once homogenized, the nuclei were recovered by centrifugation, nucleus lysis buffer was added and the sample was sheared and the chromatin fragmentation and recovery were checked.</p