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Peroxisomes play a central role in tuning metabolic and signaling programs in a tissue- and cell-type-specific manner. However, the mechanisms by which the status of peroxisomes is communicated and integrated into cellular signaling pathways are not yet understood. Herein, we report the cellular responses to peroxisomal proteotoxic stress upon silencing the peroxisomal protease/chaperone LONP2. Depletion of LONP2 triggered the accumulation of its substrate TYSND1 protease, while the overall expression of peroxisomal proteins, as well as TYSND1-dependent ACOX1 processing appeared normal, reflecting early stages of peroxisomal proteotoxic stress. Consequently, the alteration of peroxisome size and numbers, and luminal protein import failure was coupled with induction of cell-specific cellular stress responses. Specific to COS-7 cells was a strong activation of the integrated stress response (ISR) and upregulation of ribosomal biogenesis gene expression levels. Common changes between COS-7 and U2OS cell lines included repression of the retinoic acid signaling pathway and upregulation of sphingolipids. Cholesterol accumulated in the endomembrane compartments in both cell lines, consistent with evidence that peroxisomes are required for cholesterol flux out of late endosomes. These unexpected consequences of peroxisomal stress provide an important insight into our understanding of the tissue-specific responses seen in peroxisomal disorders.

In addition to fatty acids, glucose and lactate are important myocardial substrates under physiologic and stress conditions. They are metabolized to pyruvate, which enters mitochondria via the mitochondrial pyruvate carrier (MPC) for citric acid cycle metabolism. In the present study, we show that MPC-mediated mitochondrial pyruvate utilization is essential for the partitioning of glucose-derived cytosolic metabolic intermediates, which modulate myocardial stress adaptation. Mice with cardiomyocyte-restricted deletion of subunit 1 of MPC (cMPC1 −/− ) developed age-dependent pathologic cardiac hypertrophy, transitioning to a dilated cardiomyopathy and premature death. Hypertrophied hearts accumulated lactate, pyruvate and glycogen, and displayed increased protein O-linked N -acetylglucosamine, which was prevented by increasing availability of non-glucose substrates in vivo by a ketogenic diet (KD) or a high-fat diet, which reversed the structural, metabolic and functional remodelling of non-stressed cMPC1 −/− hearts. Although concurrent short-term KDs did not rescue cMPC1 −/− hearts from rapid decompensation and early mortality after pressure overload, 3 weeks of a KD before transverse aortic constriction was sufficient to rescue this phenotype. Together, our results highlight the centrality of pyruvate metabolism to myocardial metabolism and function. Metabolic substrate partitioning underlies myocardial homeostatic capacity and adaptation to stress. Zhang et al. show that decreasing mitochondrial pyruvate carrier expression redirects glycolytic intermediates and leads to heart failure, which is prevented or reversed with a high-fat or ketogenic diet.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Peroxisomes play a central role in tuning metabolic and signaling programs in a tissue- and cell type-specific manner. However, the mechanisms by which the status of peroxisomes is communicated and integrated into cellular signaling pathways is not yet understood. Herein, we report the cellular responses to acute peroxisomal proteotoxic stress upon silencing the peroxisomal protease/chaperone LONP2. Depletion of LONP2 triggered accumulation of its substrates, alterations in peroxisome size and numbers, and luminal protein import failure. Gene expression changes and lipidomic analysis revealed striking cell specific differences in the response to siLONP2. Specific to COS-7 cells was a strong activation of the integrated stress response (ISR) and upregulation of ribosomal biogenesis gene expression levels. Common changes between COS-7 and U2OS cell lines included repression of the retinoic acid signaling pathway, and upregulation of sphingolipids. Cholesterol accumulated in the endomembrane compartments in both cell lines, consistent with evidence that peroxisomes are required for cholesterol flux out of late endosomes. These unexpected consequences of peroxisomal stress provide an important insight for our understanding of the tissue-specific responses seen in peroxisomal disorders.

One of the primary metabolic functions of a mature adipocyte is to supply energy via lipolysis, or the catabolism of stored lipids. Hormone-sensitive lipase (HSL) is a critical lipolytic enzyme, and its phosphorylation and subsequent activation by PKA generates phospho-binding sites for 14-3-3 proteins, a ubiquitously expressed family of molecular scaffolds. While we previously identified essential roles of the 14-3-3ζ isoform in murine adipogenesis, the presence of 14-3-3 protein binding sites on HSL suggests that 14-3-3ζ could also influence mature adipocyte processes like lipolysis. Herein, we demonstrate that 14-3-3ζ is necessary for lipolysis in male mice and fully differentiated 3T3-L1 adipocytes, as depletion of 14-3-3ζ significantly impaired glycerol and FFA release. Unexpectedly, this was not due to impairments in signaling events underlying lipolysis; instead, reducing 14-3-3ζ expression was found to significantly impact adipocyte maturity, as observed by reduced abundance of PPARγ2 protein and expression of mature adipocytes genes and those associated with de novo triglyceride synthesis and lipolysis. The impact of 14-3-3ζ depletion on adipocyte maturity was further examined with untargeted lipidomics, which revealed that reductions in 14-3-3ζ abundance promoted the acquisition of a lipidomic signature that resembled undifferentiated, pre-adipocytes. Collectively, these findings reveal a novel aspect of 14-3-3ζ in adipocytes, as reducing 14-3-3ζ was found to have a negative effect on adipocyte maturity and adipocyte-specific processes like lipolysis.

One of the primary metabolic functions of a mature adipocyte is to supply energy via lipolysis, or the catabolism of stored lipids. Hormone-sensitive lipase (HSL) is a critical lipolytic enzyme, and its phosphorylation and subsequent activation by PKA generates phospho-binding sites for 14-3-3 proteins, a ubiquitously expressed family of molecular scaffolds. While we previously identified essential roles of the 14-3-3ζ isoform in murine adipogenesis, the presence of 14-3-3 protein binding sites on HSL suggests that 14-3-3ζ could also influence mature adipocyte processes like lipolysis. Herein, we demonstrate that 14-3-3ζ is necessary for lipolysis in male mice and fully differentiated 3T3-L1 adipocytes, as depletion of 14-3-3ζ significantly impaired glycerol and FFA release. Unexpectedly, this was not due to impairments in signaling events underlying lipolysis; instead, reducing 14-3-3ζ expression was found to significantly impact adipocyte maturity, as observed by reduced abundance of PPARγ2 protein and expression of mature adipocytes genes and those associated with de novo triglyceride synthesis and lipolysis. The impact of 14-3-3ζ depletion on adipocyte maturity was further examined with untargeted lipidomics, which revealed that reductions in 14-3-3ζ abundance promoted the acquisition of a lipidomic signature that resembled undifferentiated, pre-adipocytes. Collectively, these findings reveal a novel aspect of 14-3-3ζ in adipocytes, as reducing 14-3-3ζ was found to have a negative effect on adipocyte maturity and adipocyte-specific processes like lipolysis.

Fatty-acid (FA) signaling contributes to β-cell mass expansion in the face of nutrient excess, but the underlying mechanisms are poorly understood. Here we tested the hypothesis that sphingolipids, generated by the intracellular metabolism of FA, are implicated in the β-cell proliferative response to FA. Isolated rat islets were exposed to individual FA in the presence of 16.7 mM glucose for 48 h and the contribution of the de novo sphingolipid synthesis pathway was tested using the serine palmitoyltransferase inhibitor myriocin, the sphingosine kinase (SphK) inhibitor SKI II, or adenovirus-mediated knockdown of SphK, fatty-acid-elongase-1 (ELOVL1) and acyl-CoA-binding protein (ACBP). Wistar rat were infused with glucose and the lipid emulsion ClinOleic and received SKI II by gavage. B-cell proliferation was assessed by immunochemistry or flow cytometry. Sphingolipidomic analyses were performed by LC-MS/MS. Amongst the various FA tested, only oleate increased β-cell proliferation. Myriocin, SKI II, and SphK knockdown all decreased oleate-induced β-cell proliferation. Oleate exposure did not increase the total amount of sphingolipids but led to a specific rise in 24:1 species. Knockdown of ACBP or ELOVL1 inhibited oleate-induced β-cell proliferation. We conclude that unsaturated very long-chain sphingolipids produced from the available pool of C24:1 acyl-CoA mediate oleate-induced β-cell proliferation in rats.

La transcription des symboles et des caractères spéciaux utilisés dans la version originale de ce résumé n’a pas été possible en raison de limitations techniques. La version correcte de ce résumé peut être lue en PDF. Le contrôle du dopage sportif par les stéroïdes pouvant potentiellement se retrouver dans le corps humain de façon naturelle est très compliqué. En effet, les concentrations de ces stéroïdes peuvent varier naturellement de façon substantielle d'un individu à l'autre rendant difficile l'utilisation d'une fourchette de valeurs normales à des fins de référence. De plus, la méthode de dépistage par chromatographie en phase gazeuse et spectrométrie de masse (CG/SM) ne permet pas de différencier l'origine endogène ou exogène d'un stéroïde urinaire. C'est ici qu'entre en jeu la méthode de confirmation par la spectrométrie de masse des rapports isotopiques (CG/C/SMRI). Cette méthode, dont l'utilisation dans le domaine du contrôle de dopage sportif a été proposée en 1994, permet la différenciation des stéroïdes de sources endogène et exogène par l'analyse de leur teneur en carbone 13, isotope naturel du carbone (12C) Une première partie du projet était de vérifier la possibilité d'analyser des norstéroïdes par CG/C/SMRI. Cette analyse est limitée due aux très petites concentrations des métabolites norandrostérone (NA) et norétiocholanolone (NE) d'origine endogène retrouvées parfois dans les urines. Afin de vérifier la sensibilité de notre méthode, nous avons utilisé les urines recueillies dans le cadre d'une étude d'excrétion réalisée avec une crème à base de norandrostènedione et de norandrostènediol vendue commercialement aux États-Unis. Bien que nous ayons réussi à identifier les pics correspondants à la NA et la NE exogènes, la méthode utilisée ne nous donne pas la sensibilité requise pour l'analyse de traces. Le même problème de sensibilité est rencontré lors des analyses de rapports isotopiques de la testostérone urinaire. Généralement, sa concentration dans l'urine est trop faible pour être analysée directement par CG/C/SMRI. Nous avons comparé différentes méthodes permettant 1'isolement et la séparation par chromatographie liquide de la testostérone et de 1'épitestostérone, deux ep1meres non résolus chromatographiquement en l'absence de dérivation chimique. La méthode retenue nous permet une bonne séparation des deux stéroïdes.Le but principal du projet était de vérifier la validité de la méthode de détection de stéroïdes exogènes en réalisant une étude comparative entre trois laboratoires accrédités par L’AMA. Ces trois laboratoires, situés à Tokyo, Japon, à Cologne, Allemagne, et à Montréal, Canada, ont une longue expérience en contrôle de dopage sportif et ont chacun développé leur propre méthode de préparation des échantillons pour les analyses de rapports isotopiques. Pour réaliser 1'étude, les trois laboratoires se sont partagés des échantillons urinaires provenant de diverses études d'excrétion. Les stéroïdes étudiés lors de ces travaux sont la testostérone, la DHEA, l'androstènedione et la pregnènolone. Les résultats obtenus par les trois laboratoires sont comparés dans ce rapport non seulement en terme de valeurs absolues, mais surtout en terme de différence entre les valeurs des rapports 13C/12C des métabolites et celles des stéroïdes de référence. Ce critère est celui qui a été retenu par l'Agence mondiale antidopage afin de limiter les variations retrouvées entre les laboratoires et les variations interindividuelles. Ainsi, pour toutes les études d'excrétion, les trois laboratoires ont obtenu des résultats concordants malgré les protocoles différents, démontrant ainsi l'application universelle de la CG/C/SMRI à des fins de confirmation de 1'administration de testostérone et ses précurseurs.