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Ceramide accumulation in L6 skeletal muscle cells due to increased activity of ceramide synthase isoforms has opposing effects on insulin action to those caused by palmitate treatment

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Abstract

Aims/hypothesis

An accumulation of ceramides has been implicated in the generation of insulin resistance in skeletal muscle upon an oversupply of fatty acid. Different ceramide species are generated through the actions of ceramide synthases (CerSs), which incorporate specific acyl side chains. We tested whether particular CerS isoforms promoted insulin resistance through the generation of more inhibitory ceramide species, thus representing potential targets for intervention.

Methods

CerS isoforms CerS1, CerS2, CerS4, CerS5 and CerS6 were overexpressed in L6 myotubes using adenovirus, and cells were treated with palmitate and stimulated with insulin. Alternatively, CerS isoforms were knocked down using siRNAs. Sphingolipids were examined by mass spectrometry and tracer incorporation. Phosphorylation of IRS1 and Akt was measured by immunoblotting, while glucose disposal was assessed by measuring GLUT4 translocation and the incorporation of [14C]glucose into glycogen.

Results

Palmitate treatment increased the levels of several ceramides but reduced the levels of sphingomyelins, while insulin had no effect. The fatty acid also inhibited insulin-stimulated Akt phosphorylation and glycogen synthesis. Overexpression of CerS isoforms increased specific ceramides. Unexpectedly, the overexpression of CerS1 and CerS6 promoted insulin action, while no isoform had inhibitory effects. CerS6 knockdown had effects reciprocal to those of CerS6 overexpression.

Conclusions/interpretation

Palmitate may increase intracellular ceramide levels through sphingomyelin hydrolysis as well as de novo synthesis, but no particular species were implicated in the generation of insulin resistance. The modulation of ceramides through an alteration of CerS expression does not affect the action of insulin in the same way as ceramide generation by palmitate treatment. Conversely, certain isoforms promote insulin action, indicating the importance of ceramides in cell function.

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Fig. 1
Fig. 2

Abbreviations

CerS:

Ceramide synthase

ER:

Endoplasmic reticulum

FA:

Fatty acid

LacZ:

β-Galactosidase

siRNA:

Small interfering RNA

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Acknowledgements

The authors thank Dale Hancock (Department of Biochemistry, Sydney University, Australia) and Daniel Fazakerley (Diabetes and Obesity Program, Garvan Institute of Medical Research, Australia) for expert technical assistance, as well as Trevor Biden (Diabetes and Obesity Program, Garvan Institute of Medical Research, Australia) for critical reading of the manuscript.

Funding

This research was supported by a grant from the National Health and Medical Research Council of Australia (to CS-P), and an Australian Postgraduate Award (to BD).

Duality of interest

The authors declare that there is no duality of interest associated with this manuscript.

Contribution statement

GF, BD, B-QML, JTS, NJH, TWM and CS-P all designed and performed studies and analysed data. CS-P directed the study, interpreted the data and wrote the manuscript. All authors critically revised the manuscript and approved the final version.

Author information

Authors and Affiliations

  1. Diabetes and Obesity Program, Garvan Institute of Medical Research, 384 Victoria Street, Darlinghurst, NSW, 2010, Australia

    Georgia Frangioudakis, Barbara Diakanastasis, Bing-Qing M. Liao, Nolan J. Hoffman & Carsten Schmitz-Peiffer

  2. School of Medical Sciences, Faculty of Medicine, University of New South Wales, Sydney, NSW, Australia

    Georgia Frangioudakis

  3. School of Chemistry, University of Wollongong, Wollongong, NSW, Australia

    Jennifer T. Saville

  4. School of Health Sciences, University of Wollongong, Wollongong, NSW, Australia

    Todd W. Mitchell

  5. St Vincent’s Hospital Clinical School, Faculty of Medicine, University of New South Wales, Sydney, NSW, Australia

    Carsten Schmitz-Peiffer

Authors
  1. Georgia Frangioudakis
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  2. Barbara Diakanastasis
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  3. Bing-Qing M. Liao
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  4. Jennifer T. Saville
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  5. Nolan J. Hoffman
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  6. Todd W. Mitchell
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  7. Carsten Schmitz-Peiffer
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Corresponding author

Correspondence to Carsten Schmitz-Peiffer.

Additional information

Georgia Frangioudakis and Barbara Diakanastasis contributed equally to this work

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM Methods

(PDF 111 kb)

ESM Fig. 1

Expression of endogenous and recombinant CerS isoforms in L6 skeletal muscle cells. Myotubes were infected with recombinant adenoviruses mediating the expression of LacZ as control, or increasing amounts of the CerS isoforms as indicated (doses refer to volume of viral stock in μl). Cells were harvested and lysates subjected to immunoblotting with anti-HA antibodies (CerS1, CerS2, CerS4 and CerS5) or anti-CerS6 antibodies. (PDF 802 kb)

ESM Fig. 2

Effects of CerS isoform overexpression on minor ceramide species and on sphingomyelin species in L6 myotubes. (a) The levels of less abundant ceramide (Cer) species in basal and insulin-stimulated L6 myotubes, overexpressing the CerS isoforms indicated and pre-treated without (white bars) or with (black bars) palmitate. Results are expressed relative to Cer16:0 ceramide in basal cells infected with LacZ expressing virus and untreated with palmitate (control). ANOVA: *P < 0.05,**P < 0.01, for effect of CerS isoform vs LacZ control (± insulin, ± palmitate). (b) Myotubes infected with control adenovirus were pre-treated in the absence (white bars) or presence (black bars) of palmitate and stimulated acutely with insulin. Sphingomyelin levels in lipid extracts from cells were analysed by mass spectrometry, and expressed relative to C16:0 sphingomyelin in basal cells untreated with palmitate. D, dihydrosphingomyelin. ANOVA: ***P < 0.001 for effect of palmitate vs control (± insulin). (c) The levels of sphingomyelin species in basal and insulin-stimulated L6 myotubes, overexpressing the CerS isoforms indicated and pre-treated without (white bars) or with (black bars) palmitate. Results are expressed relative to C16:0 sphingomyelin in basal cells untreated with palmitate. ANOVA: *P < 0.05,**P < 0.01, ***P < 0.001 for effect of CerS isoform vs LacZ control (± insulin, ± palmitate). Results shown in (a-c) are the means from 3 independent experiments carried out in triplicate. (PDF 674 kb)

ESM Fig. 3

Effects of CerS isoform overexpression on mRNA levels of proteins involved in sphingolipid metabolism. RNA was extracted from L6 myotubes overexpressing CerS isoforms and pre-treated without (open bars) or with palmitate (closed bars), and real time RT-PCR performed to determine the expression of the genes indicated. Results are expressed as fold change versus cells expressing LacZ as control and are shown as means from 3 experiments carried out in quadruplicate. ANOVA: *P < 0.05 for effect of CerS1 vs LacZ on endogenous CerS1 and CerS5 expression; P < 0.05 for effect of palmitate on CerS1 expression; P < 0.001 for palmitate effect on CerS5 expression. (PDF 193 kb)

ESM Fig. 4

Effects of CerS isoform overexpression on flux through the de novo and salvage pathways of sphingolipid metabolism. L6 myotubes overexpressing CerS isoforms, or LacZ as control, and pre-treated with palmitate were labelled with either (a) [3H]serine and (b-e) [3H]sphingosine to determine flux through the de novo synthesis and salvage pathways respectively. Lipids were extracted, resolved by TLC and identified upon autoradiography using authentic standards. Lipid spots were excised and quantified by scintillation counting. Results shown are means from 3 experiments carried out in duplicate. [3H]sphingosine incorporation into different sphingolipids (b-e) is shown relative to ceramide under control conditions. ANOVA: Effect of virus on [3H]sphingosine incorporation in ceramide, P < 0.01; glucosylceramide, P < 0.05. A representative autoradiograph demonstrating [3H]sphingosine incorporation into sphingolipids is shown (f). (PDF 794 kb)

ESM Fig. 5

Effects of CerS isoform overexpression on Glut4 translocation. (a) Surface levels of HA-tagged Glut4 were measured in L6 myotubes overexpressing CerS isoforms and pre-treated without or with palmitate as indicated and stimulated without (white bars) or with (black bars) insulin. (b) Total levels of HA-tagged Glut4 were determined in permeabilised cells. Results in (a) and (b) are shown relative to levels observed in basal cells infected with LacZ control virus and not treated with palmitate. The fold increase in surface Glut4 upon insulin stimulation is shown in (c). Results shown are means from 3 experiments carried out in triplicate. ANOVA: **P < 0.001 for effect of CerS1 vs LacZ control on surface Glut4 levels under all conditions. (PDF 160 kb)

ESM Fig. 6

Validation of siRNA-mediated knockdown of CerS isoforms. (a) Differentiated L6 myotubes were transfected with 50 nM siRNA pools specifically targeting CerS1, 2, 4, 5 or 6. After 48 h, cells were harvested and CerS RNA levels quantified by RT-PCR, and expressed relative to mRNA from cells treated with control siRNA. (b) Cells transfected with CerS6-specific siRNAs were extracted and CerS6 protein detected by immunoblotting. (PDF 363 kb)

ESM Fig. 7

Effects of CerS1 and CerS6 overexpression on the respiration rate in L6 myotubes. Differentiated myotubes were infected with virus as indicated (NV, no virus) pretreated with palmitate (black bars) and stimulated with insulin (grey and black bars), prior to measurement of basal oxygen consumption rate. Results shown are means from 3 independent experiments. ANOVA: P < 0.05 for effect of palmitate vs control in insulin-stimulated cells. (PDF 108 kb)

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Frangioudakis, G., Diakanastasis, B., Liao, BQ.M. et al. Ceramide accumulation in L6 skeletal muscle cells due to increased activity of ceramide synthase isoforms has opposing effects on insulin action to those caused by palmitate treatment. Diabetologia 56, 2697–2701 (2013). https://doi.org/10.1007/s00125-013-3035-5

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  • DOI: https://doi.org/10.1007/s00125-013-3035-5

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