VLDL and apolipoprotein CIII induce ER stress and inflammation and attenuate insulin signalling via Toll-like receptor 2 in mouse skeletal muscle cells
Here, our aim was to examine whether VLDL and apolipoprotein (apo) CIII induce endoplasmic reticulum (ER) stress, inflammation and insulin resistance in skeletal muscle.
Studies were conducted in mouse C2C12 myotubes, isolated skeletal muscle and skeletal muscle from transgenic mice overexpressing apoCIII.
C2C12 myotubes exposed to VLDL showed increased levels of ER stress and inflammatory markers whereas peroxisome proliferator-activated receptor γ co-activator 1α (PGC-1α) and AMP-activated protein kinase (AMPK) levels were reduced and the insulin signalling pathway was attenuated. The effects of VLDL were also observed in isolated skeletal muscle incubated with VLDL. The changes caused by VLDL were dependent on extracellular signal-regulated kinase (ERK) 1/2 since they were prevented by the ERK1/2 inhibitor U0126 or by knockdown of this kinase by siRNA transfection. ApoCIII mimicked the effects of VLDL and its effects were also blocked by ERK1/2 inhibition, suggesting that this apolipoprotein was responsible for the effects of VLDL. Skeletal muscle from transgenic mice overexpressing apoCIII showed increased levels of some ER stress and inflammatory markers and increased phosphorylated ERK1/2 levels, whereas PGC-1α levels were reduced, confirming apoCIII effects in vivo. Finally, incubation of myotubes with a neutralising antibody against Toll-like receptor 2 abolished the effects of apoCIII on ER stress, inflammation and insulin resistance, indicating that the effects of apoCIII were mediated by this receptor.
These results imply that elevated VLDL in diabetic states can contribute to the exacerbation of insulin resistance by activating ERK1/2 through Toll-like receptor 2.
KeywordsAMPK apoCIII ERK1/2 TLR2 VLDL
AMP-activated protein kinase
- apoCIII Tg
Transgenic mice overexpressing human apoCIII
Binding immunoglobulin protein
Carnitine palmitoyltransferase 1
CCAAT-enhancer-binding protein homologous protein
Εukaryotic initiation factor 2α
Electrophoretic mobility shift assay
Extracellular signal-regulated kinase
Fatty acid oxidation
Glucose-regulated protein 78
Inhibitor of κB
IκΒ kinase β
Insulin receptor β-subunit
Inositol-requiring 1 transmembrane kinase/endonuclease-1α
Mitogen-activated protein kinase
Medium chain acyl-CoA dehydrogenase
Monocyte chemoattractant protein 1
Nuclear respiratory factor 1
Nuclear factor-E2-related factor 2
Eukaryotic translation initiation factor-2α kinase 3
Peroxisome proliferator-activated receptor γ co-activator 1α
Peroxisome proliferator-activated receptor
Suppressor of cytokine signalling 3
Signal transducer and activator of transcription 3
Unfolded protein response
X-box binding protein-1
We thank the University of Barcelona’s Language Advisory Service for revising the manuscript.
Data are available on request from the authors.
This study was partly supported by funds from the Spanish Ministerio de Economía y Competitividad (SAF2012-30708 and SAF2015-64146-R to MVC), the Generalitat de Catalunya (2014SGR-0013 to MVC), NIH NIDDK (DK101663 to ABK), USDA NIFA (11874590 to ABK) and USDA NIFA Hatch Formula Funds (2015-31200-06009 to ABK), an Instituto de Salud Carlos III grant (PI16-00139 to JCE-G) and European Union ERDF funds. CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM) is an Instituto de Salud Carlos III project (Grant CB07/08/0003 to MVC). GB was supported by an FPI grant from the Spanish Ministerio de Economía y Competitividad.
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.
All authors processed the samples, analysed and prepared the data and were involved in drafting the article. GB, AG, JCEG, XP and ABK contributed to data interpretation and revised the article. MVC designed the experiments, interpreted the data and was primarily responsible for writing the manuscript. All authors approved the final version of the manuscript. MVC is the guarantor of this work.
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