Insulinorésistance musculaire induite par les céramides : étude des mécanismes d'action et de l'implication du transporteur CERT

Obesity and type 2 diabetes are associated with a sedentary lifestyle and a diet rich in fat. Indeed, saturated fatty acids accumulate in non-adipose tissue such as skeletal muscle to generate lipids called ceramides (CER). My thesis project was divided into two parts with the objective to prevent CER to act. We have shown that, depending on the structure of the plasma membrane, CER alter the insulin signaling pathway by targeting PKB, a key insulin signalling protein, via a PKCζ pathway in L6 myotubes and a PP2A pathway in C2C12 myotubes. We also demonstrated that CER affect insulin sensitivity via the PKCζ pathway in human muscle cells. Once CER generated in the endoplasmic reticulum (ER), they are transported to the Golgi by a carrier called CERT to be metabolized into sphingomyelin (SM). Studies have shown that the transformation of CER into SM could be a crucial step to prevent CER to act. In several muscle insulin resistance models, expression of CERT is decreased and we demonstrated the importance of the transport of ceramide from the ER to the Golgi by inhibiting artificially the activity or the expression of CERT. In contrast, overexpression of CERT enhances insulin sensitivity in muscle cells in lipotoxiques conditions. Our results show that CERT plays a crucial role in mechanisms leading to the development of muscle insulin resistance since its presence is essential for maintaining normal traffic of CER between the ER and the Golgi.L'obésité et le diabète de type 2 sont associés à la sédentarité et à une alimentation riche en graisses. En effet, les acides gras saturés s'accumulent dans les tissus non adipeux, comme les muscles squelettiques pour générer des lipides appelés céramides (CER). Mon projet de thèse s'est articulé en deux parties dont l'objectif est d'empêcher les CER d'agir. Nous avons montré que, selon la structure de la membrane plasmique, les CER altèrent la voie de signalisation insulinique en ciblant la PKB, protéine clef de la voie insulinique, via la voie PKC? dans les myotubes L6 et la voie PP2A dans les myotubes C2C12. Nous avons aussi mis en évidence que les CER altèrent la sensibilité à l'insuline via la voie PKC? dans les cellules musculaires humaines. Une fois les CER produits au niveau du réticulum endoplasmique (RE), ils sont transportés au Golgi par un transporteur CERT pour y être métabolisés en sphingomyéline (SM) et des études ont montré que la transformation des CER en SM pouvait être une étape cruciale pour empêcher les CER d'agir. Dans plusieurs modèles d'insulino-résistance musculaire, l'expression de CERT est diminuée et nous avons démontré l'importance du transport des céramides du RE vers le Golgi en inhibant artificiellement l'activité ou l'expression de CERT. A l'opposé, la surexpression de CERT améliore la sensibilité à l'insuline dans les cellules musculaires dans des conditions lipotoxiques. Nos résultats montrent que CERT joue un rôle crucial dans les mécanismes conduisant au développement de l'insulinorésistance musculaire puisque sa présence est essentielle pour le maintien d'un trafic normal des CER entre le RE et le golgi

Characterising the inhibitory actions of ceramide upon insulin signaling in different skeletal muscle cell models:a mechanistic insight

International audienceCeramides are known to promote insulin resistance in a number of metabolically important tissues including skeletal muscle, the predominant site of insulin-stimulated glucose disposal. Depending on cell type, these lipid intermediates have been shown to inhibit protein kinase B (PKB/Akt), a key mediator of the metabolic actions of insulin, via two distinct pathways: one involving the action of atypical protein kinase C (aPKC) isoforms, and the second dependent on protein phosphatase-2A (PP2A). The main aim of this study was to explore the mechanisms by which ceramide inhibits PKB/Akt in three different skeletal muscle-derived cell culture models; rat L6 myotubes, mouse C2C12 myotubes and primary human skeletal muscle cells. Our findings indicate that the mechanism by which ceramide acts to repress PKB/Akt is related to the myocellular abundance of caveolin-enriched domains (CEM) present at the plasma membrane. Here, we show that ceramide-enriched-CEMs are markedly more abundant in L6 myotubes compared to C2C12 myotubes, consistent with their previously reported role in coordinating aPKC-directed repression of PKB/Akt in L6 muscle cells. In contrast, a PP2A-dependent pathway predominantly mediates ceramide-induced inhibition of PKB/Akt in C2C12 myotubes. In addition, we demonstrate for the first time that ceramide engages an aPKC-dependent pathway to suppress insulin-induced PKB/Akt activation in palmitate-treated cultured human muscle cells as well as in muscle cells from diabetic patients. Collectively, this work identifies key mechanistic differences, which may be linked to variations in plasma membrane composition, underlying the insulin-desensitising effects of ceramide in different skeletal muscle cell models that are extensively used in signal transduction and metabolic studies

Familial hypercholesterolaemia in children and adolescents from 48 countries: a cross-sectional study

Background Approximately 450 000 children are born with familial hypercholesterolaemia worldwide every year, yet only 2·1% of adults with familial hypercholesterolaemia were diagnosed before age 18 years via current diagnostic approaches, which are derived from observations in adults. We aimed to characterise children and adolescents with heterozygous familial hypercholesterolaemia (HeFH) and understand current approaches to the identification and management of familial hypercholesterolaemia to inform future public health strategies. Methods For this cross-sectional study, we assessed children and adolescents younger than 18 years with a clinical or genetic diagnosis of HeFH at the time of entry into the Familial Hypercholesterolaemia Studies Collaboration (FHSC) registry between Oct 1, 2015, and Jan 31, 2021. Data in the registry were collected from 55 regional or national registries in 48 countries. Diagnoses relying on self-reported history of familial hypercholesterolaemia and suspected secondary hypercholesterolaemia were excluded from the registry; people with untreated LDL cholesterol (LDL-C) of at least 13·0 mmol/L were excluded from this study. Data were assessed overall and by WHO region, World Bank country income status, age, diagnostic criteria, and index-case status. The main outcome of this study was to assess current identification and management of children and adolescents with familial hypercholesterolaemia. Findings Of 63 093 individuals in the FHSC registry, 11 848 (18·8%) were children or adolescents younger than 18 years with HeFH and were included in this study; 5756 (50·2%) of 11 476 included individuals were female and 5720 (49·8%) were male. Sex data were missing for 372 (3·1%) of 11 848 individuals. Median age at registry entry was 9·6 years (IQR 5·8–13·2). 10 099 (89·9%) of 11 235 included individuals had a final genetically confirmed diagnosis of familial hypercholesterolaemia and 1136 (10·1%) had a clinical diagnosis. Genetically confirmed diagnosis data or clinical diagnosis data were missing for 613 (5·2%) of 11 848 individuals. Genetic diagnosis was more common in children and adolescents from high-income countries (9427 [92·4%] of 10 202) than in children and adolescents from non-high-income countries (199 [48·0%] of 415). 3414 (31·6%) of 10 804 children or adolescents were index cases. Familial-hypercholesterolaemia-related physical signs, cardiovascular risk factors, and cardiovascular disease were uncommon, but were more common in non-high-income countries. 7557 (72·4%) of 10 428 included children or adolescents were not taking lipid-lowering medication (LLM) and had a median LDL-C of 5·00 mmol/L (IQR 4·05–6·08). Compared with genetic diagnosis, the use of unadapted clinical criteria intended for use in adults and reliant on more extreme phenotypes could result in 50–75% of children and adolescents with familial hypercholesterolaemia not being identified. Interpretation Clinical characteristics observed in adults with familial hypercholesterolaemia are uncommon in children and adolescents with familial hypercholesterolaemia, hence detection in this age group relies on measurement of LDL-C and genetic confirmation. Where genetic testing is unavailable, increased availability and use of LDL-C measurements in the first few years of life could help reduce the current gap between prevalence and detection, enabling increased use of combination LLM to reach recommended LDL-C targets early in life. Funding Pfizer, Amgen, Merck Sharp & Dohme, Sanofi–Aventis, Daiichi Sankyo, and Regeneron

Familial hypercholesterolaemia in children and adolescents from 48 countries: a cross-sectional study

Background: Approximately 450 000 children are born with familial hypercholesterolaemia worldwide every year, yet only 2·1% of adults with familial hypercholesterolaemia were diagnosed before age 18 years via current diagnostic approaches, which are derived from observations in adults. We aimed to characterise children and adolescents with heterozygous familial hypercholesterolaemia (HeFH) and understand current approaches to the identification and management of familial hypercholesterolaemia to inform future public health strategies. Methods: For this cross-sectional study, we assessed children and adolescents younger than 18 years with a clinical or genetic diagnosis of HeFH at the time of entry into the Familial Hypercholesterolaemia Studies Collaboration (FHSC) registry between Oct 1, 2015, and Jan 31, 2021. Data in the registry were collected from 55 regional or national registries in 48 countries. Diagnoses relying on self-reported history of familial hypercholesterolaemia and suspected secondary hypercholesterolaemia were excluded from the registry; people with untreated LDL cholesterol (LDL-C) of at least 13·0 mmol/L were excluded from this study. Data were assessed overall and by WHO region, World Bank country income status, age, diagnostic criteria, and index-case status. The main outcome of this study was to assess current identification and management of children and adolescents with familial hypercholesterolaemia. Findings: Of 63 093 individuals in the FHSC registry, 11 848 (18·8%) were children or adolescents younger than 18 years with HeFH and were included in this study; 5756 (50·2%) of 11 476 included individuals were female and 5720 (49·8%) were male. Sex data were missing for 372 (3·1%) of 11 848 individuals. Median age at registry entry was 9·6 years (IQR 5·8-13·2). 10 099 (89·9%) of 11 235 included individuals had a final genetically confirmed diagnosis of familial hypercholesterolaemia and 1136 (10·1%) had a clinical diagnosis. Genetically confirmed diagnosis data or clinical diagnosis data were missing for 613 (5·2%) of 11 848 individuals. Genetic diagnosis was more common in children and adolescents from high-income countries (9427 [92·4%] of 10 202) than in children and adolescents from non-high-income countries (199 [48·0%] of 415). 3414 (31·6%) of 10 804 children or adolescents were index cases. Familial-hypercholesterolaemia-related physical signs, cardiovascular risk factors, and cardiovascular disease were uncommon, but were more common in non-high-income countries. 7557 (72·4%) of 10 428 included children or adolescents were not taking lipid-lowering medication (LLM) and had a median LDL-C of 5·00 mmol/L (IQR 4·05-6·08). Compared with genetic diagnosis, the use of unadapted clinical criteria intended for use in adults and reliant on more extreme phenotypes could result in 50-75% of children and adolescents with familial hypercholesterolaemia not being identified. Interpretation: Clinical characteristics observed in adults with familial hypercholesterolaemia are uncommon in children and adolescents with familial hypercholesterolaemia, hence detection in this age group relies on measurement of LDL-C and genetic confirmation. Where genetic testing is unavailable, increased availability and use of LDL-C measurements in the first few years of life could help reduce the current gap between prevalence and detection, enabling increased use of combination LLM to reach recommended LDL-C targets early in life

Muscle insulin resistance induced by ceramide : study of the mechanism and the implication of CERT transporter

L'obésité et le diabète de type 2 sont associés à la sédentarité et à une alimentation riche en graisses. En effet, les acides gras saturés s'accumulent dans les tissus non adipeux, comme les muscles squelettiques pour générer des lipides appelés céramides (CER). Mon projet de thèse s'est articulé en deux parties dont l'objectif est d'empêcher les CER d'agir. Nous avons montré que, selon la structure de la membrane plasmique, les CER altèrent la voie de signalisation insulinique en ciblant la PKB, protéine clef de la voie insulinique, via la voie PKC? dans les myotubes L6 et la voie PP2A dans les myotubes C2C12. Nous avons aussi mis en évidence que les CER altèrent la sensibilité à l'insuline via la voie PKC? dans les cellules musculaires humaines. Une fois les CER produits au niveau du réticulum endoplasmique (RE), ils sont transportés au Golgi par un transporteur CERT pour y être métabolisés en sphingomyéline (SM) et des études ont montré que la transformation des CER en SM pouvait être une étape cruciale pour empêcher les CER d'agir. Dans plusieurs modèles d'insulino-résistance musculaire, l'expression de CERT est diminuée et nous avons démontré l'importance du transport des céramides du RE vers le Golgi en inhibant artificiellement l'activité ou l'expression de CERT. A l'opposé, la surexpression de CERT améliore la sensibilité à l'insuline dans les cellules musculaires dans des conditions lipotoxiques. Nos résultats montrent que CERT joue un rôle crucial dans les mécanismes conduisant au développement de l'insulinorésistance musculaire puisque sa présence est essentielle pour le maintien d'un trafic normal des CER entre le RE et le golgi.Obesity and type 2 diabetes are associated with a sedentary lifestyle and a diet rich in fat. Indeed, saturated fatty acids accumulate in non-adipose tissue such as skeletal muscle to generate lipids called ceramides (CER). My thesis project was divided into two parts with the objective to prevent CER to act. We have shown that, depending on the structure of the plasma membrane, CER alter the insulin signaling pathway by targeting PKB, a key insulin signalling protein, via a PKCζ pathway in L6 myotubes and a PP2A pathway in C2C12 myotubes. We also demonstrated that CER affect insulin sensitivity via the PKCζ pathway in human muscle cells. Once CER generated in the endoplasmic reticulum (ER), they are transported to the Golgi by a carrier called CERT to be metabolized into sphingomyelin (SM). Studies have shown that the transformation of CER into SM could be a crucial step to prevent CER to act. In several muscle insulin resistance models, expression of CERT is decreased and we demonstrated the importance of the transport of ceramide from the ER to the Golgi by inhibiting artificially the activity or the expression of CERT. In contrast, overexpression of CERT enhances insulin sensitivity in muscle cells in lipotoxiques conditions. Our results show that CERT plays a crucial role in mechanisms leading to the development of muscle insulin resistance since its presence is essential for maintaining normal traffic of CER between the ER and the Golgi

Targeting sphingolipid metabolism in the treatment of obesity/type 2 diabetes

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Sustained Action of Ceramide on the Insulin Signaling Pathway in Muscle Cells: IMPLICATION OF THE DOUBLE-STRANDED RNA-ACTIVATED PROTEIN KINASE*

International audienceIn vivo, ectopic accumulation of fatty acids in muscles leads to alterations in insulin signaling at both the IRS1 and Akt steps. However, in vitro treatments with saturated fatty acids or their derivative ceramide demonstrate an effect only at the Akt step. In this study, we adapted our experimental procedures to mimic the in vivo situation and show that the double-stranded RNA-dependent protein kinase (PKR) is involved in the long-term effects of saturated fatty acids on IRS1. C2C12 or human muscle cells were incubated with palmitate or directly with ceramide for short or long periods, and insulin signaling pathway activity was evaluated. PKR involvement was assessed through pharmacological and genetic studies. Short-term treatments of myotubes with palmitate, a ceramide precursor, or directly with ceramide induce an inhibition of Akt, whereas prolonged periods of treatment show an additive inhibition of insulin signaling through increased IRS1 serine 307 phosphorylation. PKR mRNA, protein, and phosphorylation are increased in insulin-resistant muscles. When PKR activity is reduced (siRNA or a pharmacological inhibitor), serine phosphorylation of IRS1 is reduced, and insulin-induced phosphorylation of Akt is improved. Finally, we show that JNK mediates ceramide-activated PKR inhibitory action on IRS1. Together, in the long term, our results show that ceramide acts at two distinct levels of the insulin signaling pathway (IRS1 and Akt). PKR, which is induced by both inflammation signals and ceramide, could play a major role in the development of insulin resistance in muscle cells

Ceramide Transporter CERT Is Involved in Muscle Insulin Signaling Defects Under Lipotoxic Conditions

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Inhibition of aPKCs sensitizes insulin-resistant human myotubes to insulin.

<p>A. Differentiated myotubes from control donors were incubated with Ro 31.8220 (5 µM, 18 h) or OKA (500 nM, last 30 min), then stimulated with 100 nM insulin for the last 10 min before being lysed. Cell lysates were immunoblotted with antibodies against Ser⁴⁷³PKB/Akt, total PKB/Akt and β-actin. B. Differentiated myotubes from diabetic patients were incubated with Ro 31.8220 (5 µM, 18 h) or OKA (500 nM, last 30 min), then stimulated with 100 nM insulin for the last 10 min before being lysed. Cell lysates were immunoblotted with antibodies against Ser⁴⁷³PKB/Akt and total PKB/Akt. Scanning densitometry was performed to quantify changes in Ser⁴⁷³PKB/Akt abundance over total PKB/Akt protein expression in cell lysates. Bars represent mean +/− SEM. * denotes significant change p<0.05 relative to the insulin treated cells. Blots represent three separate experiments.</p