ALA, EPA and DHA differentially Modulate Palmitate-induced Lipotoxicity through Alterations of its Metabolism and Storage in C12C12 Muscle Cells

On that occasion, the two French societies dedicated to lipid science and technology, GERLI and SFEL, will combine their efforts to assist the scientific committee to establish an attractive program for the Euro Fed Lipid congress.Since few decades, incidence of obesity and type 2 diabetes (T2D) is increasing. Excessive intake of energy leads to fat overload and formation of lipotoxic compounds mainly derived from the saturated fatty acid palmitate in insulin-sensitive tissues (muscle, liver and white adipose tissue), promoting insulin resistance (IR, a well-known metabolic disorder in T2D). Supplementation with n-3 fatty acids (n-3FA) is suggested to reduce lipotoxicity and IR. We hypothesized that, according to the n-3FA used, differential and specific effects on palmitate metabolism in muscle cells will be demonstrated. C2C12 myotubes were treated with 500 µM of palmitate without or with 50 µM of alpha-linolenic acid (ALA), eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) for 16 hours and collected for measurement of membrane fluidity using diphenyl-hexatriene, ceramide content, insulin-dependent Akt protein phosphorylation (as an index of IR). The assessment of the intracellular metabolism and incorporation of palmitate into lipid fractions (triglycerides, phospholipids, diglycerides) was performed after treatment for 3 hours with [1-14C]-palmitate. As expected, palmitate-induced IR was restored by EPA and DHA supplementation whereas ALA had no effect compared to palmitate alone. EPA and DHA significantly improved C2C12 membrane fluidity compared to palmitate alone (+8.5% and +13% respectively, p<0.05). Furthermore, palmitate incorporation into the diglyceride fraction was decreased by 31 and 47% by EPA and DHA vs. palmitate, respectively (p=0.05). However, DHA significantly increased the ratio of diglycerides to total lipids vs. palmitate alone (p<0.05), whereas EPA did not. Finally, EPA was more potent to decrease palmitate-induced ceramide accumulation (+174%, p<0.05 vs. control) compared to DHA (-50% and -29% repectively, p<0.05). In conclusion and contrary to ALA, EPA and DHA treatment improved the insulin signalling pathway by differently modulating membrane fluidity and lipid and palmitate metabolism, thus demonstrating that n-3FA have different metabolic impacts on C2C12 lipid metabolism

Effets comparatifs des acides gras omega-3 (ALA, EPA, DHA) sur la sensibilité à l’insuline des cellules musculaires C2C12 dans un contexte lipotoxique

Objectifs :Etudier le rôle des ω3 sur la lipotoxicité induite par l’acide gras saturé palmitate (PAL, C16:0) dans un modèle de cellule musculaire C2C12.Identifier les effets propres de chaque w3 (ALA, EPA et DHA) à dose équivalente sur la fluidité des membranes et la réponse à l’insuline.Suivre le devenir intracellulaire du [1-14C]-palmitate en présence d’un w3 et définir les classes de lipides altérées.Rechercher les voies de signalisation impliquées dans la modulation de la réponse à l’insuline

Modulation of Insulin Resistance and the Adipocyte-Skeletal Muscle Cell Cross-Talk by LCn-3PUFA

The cross-talk between skeletal muscle and adipose tissue is involved in the development of insulin resistance (IR) in skeletal muscle, leading to the decrease in the anabolic effect of insulin. We investigated if the long chain polyunsaturated n-3 fatty acids (LCn-3PUFA), eicosapentaenoic and docosapentaenoic acids (EPA and DPA, respectively) could (1) regulate the development of IR in 3T3-L1 adipocytes and C2C12 muscle cells and (2) inhibit IR in muscle cells exposed to conditioned media (CM) from insulin-resistant adipocytes. Chronic insulin (CI) treatment of adipocytes and palmitic acid (PAL) exposure of myotubes were used to induce IR in the presence, or not, of LCn-3PUFA. EPA (50 microM) and DPA (10 microM) improved PAL-induced IR in myotubes, but had only a partial effect in adipocytes. CM from adipocytes exposed to CI induced IR in C2C12 myotubes. Although DPA increased the mRNA levels of genes involved in fatty acid (FA) beta-oxidation and insulin signaling in adipocytes, it was not sufficient to reduce the secretion of inflammatory cytokines and prevent the induction of IR in myotubes exposed to adipocyte's CM. Treatment with DPA was able to increase the release of adiponectin by adipocytes into CM. In conclusion, DPA is able to protect myotubes from PAL-induced IR, but not from IR induced by CM from adipocytes

Influence of dietary eicosapentaenoic acid on the cardiac mechanical and mitochondrial functions during early sepsis

Topic 1 — Inflammation — AIntroductionSepsis triggers the failure of cardiac mitochondria and this deleterious effect could be prevented by omega-3 polyunsaturated fatty acids (PUFA).ObjectivesTo evaluate the influence of eicosapentaenoic acid (C20: 5 omega-3 or EPA) on the mechanical and mitochondrial functions of the heart during early sepsis.MethodsFour month-old female Wistar rats were subdivided in 4 groups (n = 8 per group) according to: (i) the 4-week diet given (deficient in omega-3 PUFA [DEF] or enriched with EPA); (ii) the surgery performed (caecal ligation at 1 cm of the apex of the heart and puncture in order to induce sepsis [CLP] or sham). The cardiac mechanical function was studied in vivo by NMR 48 h after the surgery. Immediately after, part of the myocardium was used in order to extract mitochondria and another part was frozen in order to subsequently analyse the fatty acid composition of cardiac phospholipids.ResultsThe EPA groups display a large decrease in the omega-6/omega-3 PUFA ratio of cardiac phospholipids. Sepsis increases the mRNA expression of IL-1beta in the DEF group but not in the EPA one. The mechanical function is altered neither by the diets nor by the surgeries. The DEF/CLP animals have a deficient oxidative phosphorylation compared to the DEF/Sham rats: reduced mitochondrial oxygen consumption (−42%, P < 0.001) and ATP production (−37%, P < 0.05). The sepsis-induced mitochondrial damages in this group is due to an increase reactive oxygen species (ROS) release (+130 and +63% for the ROS/respiration and ROS/ATP ratios, P < 0.01 and P < 0.05, respectively). The dietary EPA prevents these damages (P < 0.05).ConclusionA sufficient EPA intake favours the upholding of the mitochondrial function during early sepsis, which could delay the subsequent development of heart failure. We plan to verify this beneficial effect of EPA in Humans affected by acute endocarditis

Adipose Tissue Dysfunctions in Response to an Obesogenic Diet Are Reduced in Mice after Transgenerational Supplementation with Omega 3 Fatty Acids

International audienceObesity is characterized by profound alterations in adipose tissue (AT) biology, leading to whole body metabolic disturbances such as insulin resistance and cardiovascular diseases. These alterations are related to the development of a local inflammation, fibrosis, hypertrophy of adipocytes, and dysregulation in energy homeostasis, notably in visceral adipose tissue (VAT). Omega 3 (n-3) fatty acids (FA) have been described to possess beneficial effects against obesity-related disorders, including in the AT; however, the long-term effect across generations remains unknown. The current study was conducted to identify if supplementation with n-3 polyunsaturated FA (PUFA) for three generations could protect from the consequences of an obesogenic diet in VAT. Young mice from the third generation of a lineage receiving a daily supplementation (1% of the diet) with fish oil rich in eicosapentaenoic acid (EPA) or an isocaloric amount of sunflower oil, were fed a high-fat, high-sugar content diet for 4 months. We explore the transcriptomic adaptations in each lineage using DNA microarray in VAT and bioinformatic exploration of biological regulations using online databases. Transgenerational intake of EPA led to a reduced activation of inflammatory processes, perturbation in metabolic homeostasis, cholesterol metabolism, and mitochondrial functions in response to the obesogenic diet as compared to control mice from a control lineage. This suggests that the continuous intake of long chain n-3 PUFA could be preventive in situations of oversupply of energy-dense, nutrient-poor foods.L'obésité se caractérise par de profondes altérations de la biologie du tissu adipeux (TA), entraînant des troubles métaboliques de l'ensemble du corps tels que la résistance à l'insuline et les maladies cardiovasculaires. Ces altérations sont liées au développement d'une inflammation locale, d'une fibrose, d'une hypertrophie des adipocytes et d'un dérèglement de l'homéostasie énergétique, notamment du tissu adipeux viscéral (TVA). Les acides gras oméga 3 (n-3) (AF) ont été décrits comme possédant des effets bénéfiques contre les troubles liés à l'obésité, y compris dans l'AT ; cependant, l'effet à long terme à travers les générations reste inconnu. La présente étude a été menée pour identifier si une supplémentation en AG polyinsaturés n-3 (AGPI) pendant trois générations pouvait protéger des conséquences d'un régime obésogène dans le VAT. Des jeunes souris de la troisième génération d'une lignée recevant une supplémentation quotidienne (1% de l'alimentation) avec de l'huile de poisson riche en acide eicosapentaénoïque (EPA) ou une quantité isocalorique d'huile de tournesol, ont été nourries avec une alimentation riche en graisses et en sucre. pendant 4 mois. Nous explorons les adaptations transcriptomiques dans chaque lignée à l'aide de puces à ADN dans VAT et l'exploration bioinformatique des régulations biologiques à l'aide de bases de données en ligne. L'apport transgénérationnel d'EPA a conduit à une activation réduite des processus inflammatoires, une perturbation de l'homéostasie métabolique, du métabolisme du cholestérol et des fonctions mitochondriales en réponse au régime obésogène par rapport aux souris témoins d'une lignée témoin. Cela suggère que l'apport continu d'AGPI n-3 à longue chaîne pourrait être préventif dans les situations d'offre excédentaire d'aliments riches en énergie et pauvres en nutriments. ont été nourris avec un régime riche en graisses et en sucre pendant 4 mois. Nous explorons les adaptations transcriptomiques dans chaque lignée à l'aide de puces à ADN dans VAT et l'exploration bioinformatique des régulations biologiques à l'aide de bases de données en ligne. L'apport transgénérationnel d'EPA a conduit à une activation réduite des processus inflammatoires, une perturbation de l'homéostasie métabolique, du métabolisme du cholestérol et des fonctions mitochondriales en réponse au régime obésogène par rapport aux souris témoins d'une lignée témoin. Cela suggère que l'apport continu d'AGPI n-3 à longue chaîne pourrait être préventif dans les situations d'offre excédentaire d'aliments riches en énergie et pauvres en nutriments. ont été nourris avec un régime riche en graisses et en sucre pendant 4 mois. Nous explorons les adaptations transcriptomiques dans chaque lignée à l'aide de puces à ADN dans VAT et l'exploration bioinformatique des régulations biologiques à l'aide de bases de données en ligne. L'apport transgénérationnel d'EPA a conduit à une activation réduite des processus inflammatoires, une perturbation de l'homéostasie métabolique, du métabolisme du cholestérol et des fonctions mitochondriales en réponse au régime obésogène par rapport aux souris témoins d'une lignée témoin. Cela suggère que l'apport continu d'AGPI n-3 à longue chaîne pourrait être préventif dans les situations d'offre excédentaire d'aliments riches en énergie et pauvres en nutriments. L'apport transgénérationnel d'EPA a conduit à une activation réduite des processus inflammatoires, une perturbation de l'homéostasie métabolique, du métabolisme du cholestérol et des fonctions mitochondriales en réponse au régime obésogène par rapport aux souris témoins d'une lignée témoin. Cela suggère que l'apport continu d'AGPI n-3 à longue chaîne pourrait être préventif dans les situations d'offre excédentaire d'aliments riches en énergie et pauvres en nutriments. L'apport transgénérationnel d'EPA a conduit à une activation réduite des processus inflammatoires, une perturbation de l'homéostasie métabolique, du métabolisme du cholestérol et des fonctions mitochondriales en réponse au régime obésogène par rapport aux souris témoins d'une lignée témoin. Cela suggère que l'apport continu d'AGPI n-3 à longue chaîne pourrait être préventif dans les situations d'offre excédentaire d'aliments riches en énergie et pauvres en nutriments

Modulation of Insulin Resistance and the Adipocyte-Skeletal Muscle Cell Cross-Talk by LCn-3PUFA

The cross-talk between skeletal muscle and adipose tissue is involved in the development of insulin resistance (IR) in skeletal muscle, leading to the decrease in the anabolic effect of insulin. We investigated if the long chain polyunsaturated n-3 fatty acids (LCn-3PUFA), eicosapentaenoic and docosapentaenoic acids (EPA and DPA, respectively) could (1) regulate the development of IR in 3T3-L1 adipocytes and C2C12 muscle cells and (2) inhibit IR in muscle cells exposed to conditioned media (CM) from insulin-resistant adipocytes. Chronic insulin (CI) treatment of adipocytes and palmitic acid (PAL) exposure of myotubes were used to induce IR in the presence, or not, of LCn-3PUFA. EPA (50 microM) and DPA (10 microM) improved PAL-induced IR in myotubes, but had only a partial effect in adipocytes. CM from adipocytes exposed to CI induced IR in C2C12 myotubes. Although DPA increased the mRNA levels of genes involved in fatty acid (FA) beta-oxidation and insulin signaling in adipocytes, it was not sufficient to reduce the secretion of inflammatory cytokines and prevent the induction of IR in myotubes exposed to adipocyte's CM. Treatment with DPA was able to increase the release of adiponectin by adipocytes into CM. In conclusion, DPA is able to protect myotubes from PAL-induced IR, but not from IR induced by CM from adipocytes

Does Increased Abdominal obesity Depress Cardiac Function through a Change in Phospholipid Fatty Acid Composition?

National audienceBackground: High fat diets increase abdominal obesity, which is known to induce cardiomyopathy in the long term. The purpose of this study was to determine the mechanism of high abdominal adiposity-induced changes in cardiac function. Methods: Rats were fed a control diet (5% of equilibrated lipids) or a lard-enriched one (LD, 54% of lipids) for 3 months from the age of 3 months old. The fatty acid composition of cardiac phospholipids was determined and the in vivo and ex vivo cardiac function as well as ex vivo coronary reactivity were determined. The results were completed by measurements of cytosolic redox potential and mitochondrial oxidative stress. Results: LD stimulated elongases and inhibited D5- and D6- desaturases, which resulted in decreased 18:2n-6 and increased 20:4n-6 proportions in cardiac phospholipids. The augmented proportion of 20:4n-6 was responsible for an increased ex vivo coronary reactivity, since cyclooxygenase inhibition suppressed the difference. It could also explain the augmented in vivo cardiac function. However, when measured ex vivo, the cardiac function was depressed by the LD and this was associated with reduced glycolytic rate and cytosolic redox potential as well as mitochondrial oxidative stress. Conclusion: To respond to the increased energy needs related to obesity, the animals augmented their cardiac function through an accumulation of arachidonic acid. However, high fat-induced disturbances of the glycolytic pathway diminished reduced equivalents available for the glutathione peroxidase and increased the mitochondrial oxidative stress. Mitochondrial oxidative stress is known to inhibit the aconitase enzyme of the Krebs cycle and to reduce the energy production. The energy disequilibrium observed in that situation is thus probably responsible for the later cardiomyopathy occurring with type-2 diabetes

Role of p38MAPK in palmitate-induced inflammation in C2C12 muscle cells

The saturated fatty acid palmitate (PAL) is now recognized as an inducer of inflammation in many types of cells, including adipocytes and muscle cells, whereas n-3 polyunsaturated fatty acids (n-3PUFA) have anti-inflammatory properties. Meanwhile, PAL induces muscle insulin resistance (IR) and n-3PUFA are suggested to reverse it. We investigated n-3PUFA effects on palmitate-induced inflammation and the potential link between inflammation, P38MAPK activation and IR in C2C12 myotubes. After 16 hours incubation with 500µM PAL without or with SB203580, a specific inhibitor of p38MAPK, 50µM of alpha linolenic acid (ALA), eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA), myotubes were harvested and submitted to mRNA quantification or immunoblotting. IL-6, TNF-a and COX-2 mRNA levels were significantly increased by PAL treatment and reversed by EPA and DHA. Inhibition of p38MAPK significantly prevented the effect of PAL on these regulations although it had no significant effect on PAL-induced IR. Additional study should be performed to explore the involvement of NFKB signalling. Our results suggested that p38MAPK activation by PAL is crucial to induce inflammation in C212 muscle cells, independently of IR. Among n-3PUFA, only EPA and DHA reduced p38MAPK activation and improved IR. Additional studies are currently performed to explore the involvement of NFKB signalling in the effect of PAL on myotube inflammation and the impact of n-3 PUFA on this pathwa