Evaluation of Mitochondrial Functions and Dysfunctions in Muscle Biopsy Samples

Preface VII Part 3 Muscle Biopsy: Metabolic Diseases 83 Chapter 5Evaluation of mitochondrial functions and dysfunctions in muscle biopsy sample

E2F transcription factor-1 modulates expression of glutamine metabolic genes in mouse embryonic fibroblasts and uterine sarcoma cells

Metabolic reprogramming is considered as a hallmark of cancer and is clinically exploited as a novel target for therapy. The E2F transcription factor-1 (E2F1) regulates various cellular processes, including proliferative and metabolic pathways, and acts, depending on the cellular and molecular context, as an oncogene or tumor suppressor. The latter is evident by the observation that E2f1-knockout mice develop spontaneous tumors, including uterine sarcomas. This dual role warrants a detailed investigation of how E2F1 loss impacts metabolic pathways related to cancer progression. Our data indicate that E2F1 binds to the promoter of several glutamine metabolism-related genes. Interestingly, the expression of genes in the glutamine metabolic pathway were increased in mouse embryonic fibroblasts (MEFs) lacking E2F1. In addition, we confirm that E2f1 <sup>-/-</sup> MEFs are more efficient in metabolizing glutamine and producing glutamine-derived precursors for proliferation. Mechanistically, we observe a co-occupancy of E2F1 and MYC on glutamine metabolic promoters, increased MYC binding after E2F1 depletion and that silencing of MYC decreased the expression of glutamine-related genes in E2f1 <sup>-/-</sup> MEFs. Analyses of transcriptomic profiles in 29 different human cancers identified uterine sarcoma that showed a negative correlation between E2F1 and glutamine metabolic genes. CRISPR/Cas9 knockout of E2F1 in the uterine sarcoma cell line SK-UT-1 confirmed elevated glutamine metabolic gene expression, increased proliferation and increased MYC binding to glutamine-related promoters upon E2F1 loss. Together, our data suggest a crucial role of E2F1 in energy metabolism and metabolic adaptation in uterine sarcoma cells

Fonctions mitochondriales et étiologie de la résistance à l'insuline dans le muscle : approches chez l'homme et sur modèles animaux

Mitochondrial functions in the aetiology of muscle insulin resistance : clinical and animal studies. Altered mitochondrial oxidative capacities may be involved in the aetiology of muscle insulin resistance, thus promoting onset and development of type 2 diabetes. This work aimed at determining how obesity, high fat diet and oxidative stress respectively influence skeletal muscle insulin sensitivity and mitochondrial function. In a human study, we found that mild obesity is characterized by normal insulin sensitivity and a raise in mitochondrial respiration, whereas severe obesity is linked to insulin resistance and decreased mitochondrial respiration and ATP production. In Wistar rats fed a high fat diet for 14 days, insulin sensitivity was not altered while mitochondrial function was stimulated. Extending high fat diet to 40 days is accompanied by a decline in insulin sensitivity concomitantly wiht decreased mitochondrial respiration. In twelve-month-old SAM mice, exhibiting spontaneous chronic oxidative stress, mitochondrial respiration and muscle insulin sensitivity are stimulated compared to control mice. In the three models described, mitochondrial function modifications occur without any change in muscle mitochondrial content. In conclusion, this work evidenced that mitochondrial function adapts intrinsically to environmental conditions associated with the pathogenesis of type 2 diabetes. Following energy excess, mitochondrial function is first stimulated which may allow protecting from insulin resistance, potentially through a mild and beneficial oxidative stress. Thereafter, when obesity increases or high fat diet is extended, other regulatory pathways take place and lead to simultaneous alterations in insulin sensitivity and mitochondrial function.L'altération des fonctions mitochondriales (FM) pourrait être impliquée dans l'étiologie de la résistance à l'insuline musculaire, et donc du diabète de type 2. Ce travail avait pour objectif de déterminer l'influence de l'obésité, d'un régime hyperlipidique (HL) ou d'un stress oxydant chronique (SO) sur la sensibilité à l'insuline et les FM dans le muscle squelettique. Chez l'Homme, l'obésité modérée s'accompagne d'un maintien de la sensibilité à l'insuline associé à une augmentation de la respiration mitochondriale (RM), tandis qu'une obésité accrue est caractérisée par une insulino-résistance et par la réduction de la RM et de la production d'ATP. Chez le rat Wistar soumis à un régime HL, la sensibilité à l'insuline est maintenue et la RM est augmentée après 14 jours, alors qu'après 40 jours s'installe une insulino-résistance accompagnée d'une diminution de la RM. Chez la souris SAM, qui développe spontanément un SO, la RM est stimulée et la sensibilité à l'insuline musculaire améliorée. Dans ces trois modèles, les modifications des FM ont été observées en l'absence de modification du contenu en mitochondries. En conclusion, ce travail montre que les FM présentent des adaptations intrinsèques dans différentes situations associées à la physiopathologie du diabète de type 2. En réponse à un excès énergétique, les FM sont dans un premier temps stimulées, ce qui pourrait participer au maintien de la sensibilité à l'insuline, potentiellement au travers d'un stress oxydant modéré. Lorsque l'obésité est aggravée ou le régime prolongé, d'autres mécanismes de régulation interviennent aboutissant à l'altération simultanée de la sensibilité à l'insuline et des FM

Skeletal muscle insulin resistance physiopathology and mitochondrial function

National audienceIn modern societies, decreased physical activity, nutritional transition and aging contribute to the increase in the prevalence of obesity and its associated pathologies (as cardiovascular diseases and type 2 diabetes). Obesity is tightly correlated with insulin resistance, which appears in the early stages of type 2 diabetes. As skeletal muscle is quantitatively the main tissue involved in glucose transport in response to insulin, muscle insulin resistance is a key step in the etiology of type 2 diabetes. Several alterations of skeletal muscle insulin signaling in various models of obesity or type 2 diabetes have been evidenced and numerous underlying mechanisms have been hypothesized. Among them, muscle lipotoxicity, obesity-induced adipose tissue inflammation and oxidative stress following excess of energetic substrates could be involved, independently or synergically, in the development of muscle insulin resistance. Moreover, mitochondrial alterations have been reported in the skeletal muscle of obese or diabetic patients and animals. This suggests that mitochondrion, through its capacity of regulating cellular fatty acids flux and redox state, may play a key role in obesity-induced skeletal muscle insulin resistance. (C) 2011 Elsevier Masson SAS. All rights reserved

Fonctions mitochondriales et étiologie de la résistance à l'insuline dans le muscle (approches chez l'homme et sur modèles animaux)

L'altération des fonctions mitochondriales (FM) pourrait être impliquée dans l'étiologie de la résistance à l'insuline musculaire, et donc du diabète de type 2. Ce travail avait pour objectif de déterminer l'influence de l'obésité, d'un régime hyperlipidique (HL) ou d'un stress oxydant chronique (SO) sur la sensibilité à l'insuline et les FM dans le muscle squelettique. Chez l'Homme, l'obésité modérée s'accompagne d'un maintien de la sensibilité à l'insuline associé à une augmentation de la respiration mitochondriale (RM), tandis qu'une obésité accrue est caractérisée par une insulino-résistance et par la réduction de la RM et de la production d'ATP. Chez le rat Wistar soumis à un régime HL, la sensibilité à l'insuline est maintenue et la RM est augmentée après 14 jours, alors qu'après 40 jours s'installe une insulino-résistance accompagnée d'une diminution de la RM. Chez la souris SAM, qui développe spontanément un SO, la RM est stimulée et la sensibilité à l'insuline musculaire améliorée. Dans ces trois modèles, les modifications des FM ont été observées en l'absence de modification du contenu en mitochondries. En conclusion, ce travail montre que les FM présentent des adaptations intrinsèques dans différentes situations associées à la physiopathologie du diabète de type 2. En réponse à un excès énergétique, les FM sont dans un premier temps stimulées, ce qui pourrait participer au maintien de la sensibilité à l'insuline, potentiellement au travers d'un stress oxydant modéré. Lorsque l'obésité est aggravée ou le régime prolongé, d'autres mécanismes de régulation interviennent aboutissant à l'altération simultanée de la sensibilité à l'insuline et des FM.CLERMONT FD-BCIU-Santé (631132104) / SudocSudocFranceF

Control of adipogenesis by oxylipins, GPCRs and PPARs

International audienceOxylipins are bioactive metabolites derived from the oxygenation of ω3 and ω6 polyunsaturated fatty acids, triggered essentially by cyclooxygenase and lipoxygenase activities. Oxylipins are involved in the development and function of adipose tissue and their productions are strictly related to diet quality and quantity. Oxylipins signal via cell surface membrane (G Protein-coupled receptors) and nuclear receptors (peroxisome proliferator-activated receptors), two pathways playing a pivotal role in adipocyte biology. In this review, we made an attempt to cover the available knowledge about synthesis and molecular function of oxylipins known to modulate adipogenesis, adipocyte function and phenotype conversion, with a focus on their interaction with peroxisome proliferator-activated nuclear receptor family

In vitro brown and "brite"/"beige" adipogenesis: Human cellular models and molecular aspects.

International audienceBrown adipose tissue (BAT) has long been thought to be absent or very scarce in human adults so that its contribution to energy expenditure was not considered as relevant. The recent discovery of thermogenic BAT in human adults opened the field for innovative strategies to combat overweight/obesity and associated diseases. This energy-dissipating function of BAT is responsible for adaptive thermogenesis in response to cold stimulation. In this context, adipocytes can be converted, within white adipose tissue (WAT), into multilocular adipocytes expressing UCP1, a mitochondrial protein that plays a key role in heat production by uncoupling the activity of the respiratory chain from ATP synthesis. These adipocytes have been named "brite" or "beige" adipocytes. Whereas BAT has been studied for a long time in murine models both in vivo and in vitro, there is now a strong demand for human cellular models to validate and/or identify critical factors involved in the induction of a thermogenic program within adipocytes. In this review we will discuss the different human cellular models described in the literature and what is known regarding the regulation of their differentiation and/or activation process. In addition, the role of microRNAs as novel regulators of brown/"brite" adipocyte differentiation and conversion will be depicted. Finally, investigation of both the conversion and the metabolism of white-to-brown converted adipocytes is required for the development of therapeutic strategies targeting overweight/obesity and associated diseases. This article is part of a Special Issue entitled Brown and White Fat: From Signaling to Disease

Method for functional study of mitochondria in rat hypothalamus

1872-678X (Electronic) Journal Article Research Support, Non-U.S. Gov'tDifferent roles of mitochondria in brain function according to brain area are now clearly emerging. Unfortunately, no technique is yet described to investigate mitochondria function in specific brain area. In this article, we provide a complete description of a procedure to analyze the mitochondrial function in rat brain biopsies. Our two-step method consists in a saponin permeabilization of fresh brain tissues in combination with high-resolution respirometry to acquire the integrated respiratory rate of the biopsy. In the first part, we carefully checked the mitochondria integrity after permeabilization, defined experimental conditions to determine the respiratory control ratio (RCR), and tested the reproducibility of this technique. In the second part, we applied our method to test its sensitivity. As a result, this method was sensitive enough to reveal region specificity of mitochondrial respiration within the brain. Moreover, we detected physiopathological modulation of the mitochondrial function in the hypothalamus. Thus this new technique that takes all cell types into account, and does not discard or select any mitochondria sub-population is very suitable to analyze the integrated mitochondrial respiration of brain biopsies

Reactive oxygen species enhance mitochondrial function, insulin sensitivity and glucose uptake in skeletal muscle of senescence accelerated prone mice SAMP8

Whereas reactive oxygen species (ROS) can have opposite impacts on insulin signaling, they have mainlybeen associated with mitochondrial dysfunction in skeletal muscle. We analyzed the relationship betweenthese three features in skeletal muscle of senescence accelerated mice (SAM) prone (P8), which arecharacterized by enhanced oxidative stress compared to SAM resistant (R1). Oxidative stress, ROSproduction, antioxidant system, mitochondrial content and functioning, as well as in vitro and in vivo insulinsignaling were investigated in gastrocnemius and quadriceps muscles. In SAMP8 compared to SAMR1,muscle content in carbonylated proteins was two-fold (p<0.01) and ROS production by xanthine oxidase70% (p<0.05) higher. Furthermore, insulin-induced Akt phosphorylation measured in vivo and ex vivo aswell as muscle glucose uptake measured ex vivo were significantly higher (p<0.05). Mitochondrialrespiration evidenced uncoupling and higher respiration rates with substrates of complexes II and IV, inagreement with higher maximal activity of complexes II and IV (+18 and 62%, respectively, p<0.05). Bycontrast, maximal activity of complex I was 22% lower (p<0.05). All strain differences were corrected after6 months of N-acetylcysteine (NAC) treatment, thus supporting the involvement of high ROS production inthese differences. In conclusion in muscle of SAMP8 compared to SAMR1, high ROS production isassociated to higher insulin sensitivity and glucose uptake but to lower mitochondrial complex I activity.These conflicting adaptations, with regards to the resulting imbalance between NADH production and use,were associated with intrinsic adjustments in the mitochondrial respiration chain (mitochondrial uncoupling,enhanced complexes II and IV activity). We propose that these bioenergetics adaptations may help atpreserving muscle metabolic flexibility of SAMP8

Un stress oxydant chronique est associé à une amelioration de la sensibilité à l’insuline et des fonctions mitochondrialesdans le muscle squelettique de souris SAMP8

National audienceDe nombreuses études suggèrent que les espèces réactives del’oxygène participent à l’étiologie du diabète de type 2. Ce travail a pour objectifd’étudier l’effet d’un stress oxydant chronique sur le développement de l’insulino-résistance et sur les adaptations des fonctions mitochondriales musculaires