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

Trauma exposure and PTSD prevalence among Yazidi, Christian and Muslim asylum seekers and refugees displaced to Iraqi Kurdistan

International audienceBackground: There is unreliable, and negligible information on the mental health and trauma-exposure of asylum-seekers and displaced refugees in the Iraqi Kurdistan region.Objectives: To evaluate how responsible the ethno-religious origins are, for the prevalence of trauma exposure and post-traumatic stress disorder (PTSD) in displaced Iraqi asylum-seekers and refugees residing in the Iraqi Kurdistan region.Methods: Structured interviews with a cross-sectional sample of 150 individuals, comprised of three self-identified ethno-religious groups (50 participants in each): Christians, Muslims, and Yazidis.Results: 100% prevalence of trauma exposure and 48.7% of current PTSD among refugees, 70% PTSD rate of Yazidi participants, which is significantly higher (p < 0.01) compared to 44% of Muslim participants and 32% of Christian participants. These findings were corroborated using the self-rated PTSD, DSM-5 Checklist, with more severe PTSD symptom scores (p < 0.001) obtained among Yazidis (43.1; 19.7), compared to Muslims (31.3; 20.1) and Christians (29.3; 17.8). Self-rated depressive symptoms (Patient Health Questionnaire-9) were also higher (p < 0.007) among Yazidis (12.3; 8.2) and Muslims (11.7; 5.9), compared to Christians (8.1; 7)

Effect of Palmitate on ceramide production and insulin sensitivity in human muscle cells.

<p>Human myotubes were incubated with 0.75 µM myriocin. A. Following this incubation, muscle cells were harvested in ice-cold PBS and lipids extracted to assess ceramide species content as described in the Methods section. Bars represent mean ± SEM from 3 separate experiments and * denote a significant difference from the untreated control values (P<0.05). B. Cells were then stimulated with 100 nM insulin for the last 10 min before being lysed and immunoblotted using either a phospho-specific antibody directed against Ser⁴⁷³PKB/Akt or a pan PKB antibody.</p

Tecovirimat is highly efficient on the Monkeypox virus lineage responsible for the international 2022 outbreak

The ongoing monkeypox virus (MPXV) outbreak is the largest ever recorded outside of Africa. Genomic analysis revealed a divergent phylogenetic lineage within clade 3, and atypical clinical presentations have been noted. We report the sequencing and isolation of the virus from the first clinical case diagnosed in France in May 2022. We tested the in vitro effect of tecovirimat (ST-246), a FDA approved drug, against this novel strain, showing efficacy at the nanomolar range. In comparison, cidofovir showed activity at micromolar concentrations. These results and the safety profile of tecovirimat strongly support its use in clinical care of severe forms for the 2022 MPXV outbreak

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

Levels of caveolin expression and ceramide content in both L6 and C2C12 myotubes.

<p>A. 15 µg of L6 and C2C12 lysates were immunoblotted alongside 65 ng and 130 ng of recombinant caveolin-3-GST (cav-3-GST) protein with an antibody against caveolin-3 (left panel). Bands were quantified and compared to known quantities of recombinant cav-3 proteins. Results were expressed as ng of cav-3 protein per µg of L6 or C2C12 protein lysat (right panel). B. L6 and C2C12 myotubes were solubilized in 1% Triton X-100 at 4°C and fractionated on sucrose gradients to isolate detergent resistant membranes (DRM) as described in the Methods section. Resulting fractions were collected from top to bottom of the gradient. Equal amounts of protein (1 µg) from fractions 2 to 8 of the sucrose gradient were then immunoblotted using an anti-caveolin-3 antibody. Bands were quantified and caveolin-3 content in L6 cells was expressed as fold increase relative to caveolin-3 content in C2C12 cells. * Significant change p<0.05 relative to C2C12 cells. These are representative of at least three independent experiments. C. Total ceramide content was quantified in DRM-containing fractions (3–8 of the sucrose gradient) from both L6 and C2C12 myotubes. These are representative of three independent experiments.</p

Mechanism of ceramide action on the insulin signalling pathway in both L6 and C2C12 myotubes.

<p>A. L6 and C2C12 myotubes were treated with 100 µM C2-ceramide for 2 h before isolation of DRMs. Equal amounts of protein (1 µg) of each fraction were then immunoblotted for the presence of PKCζ, PP2A and caveolin-3. These are representative immunoblots from three independent experiments. B. L6 myotubes were pre-incubated with 100 µM C2-ceramide for 2 h and with 5 mM MβCD for the last 30 min. Cells were then stimulated with 100 nM insulin for 10 min before being lysed and immunoblotted using either a phospho-specific antibody directed against Ser⁴⁷³PKB/Akt or a pan PKB antibody. C. Control C2C12 myotubes or KD-PKCζ-infected C2C12 myotubes were incubated with 100 µM C2-ceramide for 2 h or with 0.75 mM palmitate for 16 h. Control C2C12 myotubes were then treated with 500 nM okadaic acid (OKA) or with 5 mM MβCD the last 30 min. All cells were treated with 100 nM insulin for the last 10 min before being lysed. Cell lysates were immunoblotted with antibodies against native PKB/Akt, Ser⁴⁷³PKB/Akt, Ser^21/9GSK3 α/β, β-actin and hemagglutinin (HA). Scanning densitometry was performed to quantify changes in Ser⁴⁷³PKB/Akt abundance in cell lysates. Bars represent mean +/− SEM. * Significant change p<0.05 relative to the untreated control. Blots shown represent at least three separate experiments.</p