VLDL and apolipoprotein CIII induce ER stress and inflammation and attenuate insulin signalling via Toll-like receptor 2 in mouse skeletal muscle cells
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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
Insulin resistance and type 2 diabetes mellitus are characterised by the presence of atherogenic dyslipidaemia, which includes the following cluster of abnormalities: high levels of triacylglycerols, low levels of HDL-cholesterol and the appearance of small, dense LDLs . Atherogenic dyslipidaemia frequently precedes type 2 diabetes mellitus by several years, indicating that derangement of lipid metabolism is an early event in the development of this disease . It is now well accepted that the different components of atherogenic dyslipidaemia are closely linked and are initiated by insulin resistance through overproduction of triacylglycerol-rich VLDL [1, 2]. In addition to triacylglycerols, VLDLs also contain apolipoproteins, of which apolipoprotein (apo) CIII is one of the most abundant  with levels that are closely correlated with serum triacylglycerol levels . Plasma apoCIII increases plasma triacylglycerols predominantly through the inhibition of VLDL hydrolysis by lipoprotein lipase and by inhibiting chylomicron and VLDL clearance by the liver , but it also causes inflammation in endothelial cells . Furthermore, some studies have associated elevated circulating apoCIII with insulin resistance , although others did not find a relationship .
Whereas the effects of insulin resistance on lipoprotein metabolism have been studied extensively [1, 2], little is known about the effects of elevated VLDL and apoCIII on the molecular mechanism of insulin resistance in skeletal muscle cells. This is important, since the primary site of insulin-stimulated glucose disposal is skeletal muscle and this can account for up to 90% of glucose clearance . As a result, loss of skeletal muscle insulin sensitivity is believed to be critical in the pathogenesis of type 2 diabetes . The mechanisms involved in the development of insulin resistance are currently unclear, but accumulating evidence points to the presence of a chronic low-level inflammatory process . Among other mechanisms, endoplasmic reticulum (ER) stress  and Toll-like receptors (TLRs)  can activate proinflammatory signalling pathways, including inhibitor of κB (IκΒ) kinase β (ΙΚΚ-β)–NF-κB. Thus, IKK-β phosphorylates IRS-1 on serine residues, attenuating the insulin signalling pathway whereas, once activated, NF-κB regulates the expression of multiple inflammatory mediators, which also contribute to insulin resistance .
In the present study, we examined whether VLDL and apoCIII induce ER stress, inflammation and insulin resistance in skeletal muscle cells.
Escherichia coli (K12 strain) lipopolysaccharide (ultrapure) and PAM3CSK4 (tripalmitoylated cysteine-, serine- and lysine-containing peptide) were purchased from InvivoGen (San Diego, CA, USA). LDH Cytotoxicity Assay Kit (88953) was from Thermo Scientific (Waltham, MA, USA) and the Elisa kit for measuring IL-6 secretion (Novex, KMC0061) was from Life Technologies (Carlsbad, CA, USA).
Plasma VLDL isolation
VLDL particles (< 1.006 g/ml) were isolated by ultracentrifugation at 100,000g for 24 h from normolipidaemic human plasma obtained in EDTA-containing vacutainer tubes (total cholesterol ≤ 5.2 mmol/l, triacylglycerols ≤ 1 mmol/l). To obtain VLDL particles containing low or high amounts of apoCIII, we further isolated light VLDL (Svedberg flotation units 60–400) from normolipidaemic and hypertriacylglycerolaemic (triacylglycerols ≥ 2.5 mmol/l) human plasma by ultracentrifugation at 56,000g for 1 h. VLDL preparations were extensively dialysed in PBS and then triacylglycerol and apoB concentrations were measured using a commercial kit adapted to a COBAS c501 autoanalyser (Roche Diagnostics, Rotkreuz, Switzerland). ApoB/triacylglycerol ratios were similar in both light VLDL preparations. ApoCIII levels were determined using a nephelometric commercial kit (Kamiya Biomedical Company, Seattle, WA, USA) adapted to COBAS c501 autoanalyser. Cells were treated with 300 μg/ml of filtered VLDL, based on triacylglycerol concentration, as previously described .
Mouse mycoplasma free C2C12 cells (ATCC, Manassas, VA, USA) were maintained, grown and differentiated to myotubes as previously described . ATCC provided authentication of the cells. Where indicated, cells were treated with 10 μmol/l U0126, 100 μg/ml apoCIII (purity > 95%) (Abcam, Cambridge, UK), 50 μg/ml TLR2 neutralising antibody (InvivoGen) or control non-immune IgG for 24 h. Cells were transiently transfected with 50 nmol/l siRNA against extracellular signal-regulated kinase (ERK) 1/2 (Santa Cruz, Dallas, TX, USA) and siRNA control using Lipofectamine 2000 (Life Technologies) according to the manufacturer’s instructions.
Skeletal muscle (gastrocnemius) samples from male wild-type and transgenic mice overexpressing human apoCIII (apoCIII Tg; C57BL/6J background) were frozen in liquid nitrogen and then stored at − 80°C. For ex vivo experiments, skeletal muscles were isolated from male C57BL/6J mice (6–8 weeks old) and mounted in an incubation bath as previously described  in the presence or absence of 500 μg/ml VLDL. Experimenters were not blind to group assignment or outcome assessment. For further details, please refer to the electronic supplementary material (ESM) Methods.
RNA preparation and quantitative RT-PCR
Isolation of total and nuclear protein extracts was performed as described elsewhere . Western blot analysis was performed using antibodies against total (1:1000, 9272) and phospho-Akt (Ser473) (1:1000, 9271), glucose-regulated protein78 (GRP78)/binding immunoglobulin protein (BiP) (1:1000, 3177), insulin receptor β-subunit (IRβ) (1:1000, 3020), CCAT-enhancer-binding protein homologous protein (CHOP) (1:1000, 5554), total eukaryotic initiation factor 2α (eIF2α) (1:1000, 9722) and phospho-eIF2α (Ser51) (1:1000, 9721S), total signal transducer and activator of transcription 3 (STAT3) (1:1000, 9132) and phospho-STAT3 (Tyr705) (1:1000, 9131), total extracellular signal-regulated kinase (ERK) 1/2 (1:1000, 9102) and phospho-ERK1/2 (Thr202/Tyr204) (1:1000, 9101), total acetyl-CoA carboxylase (ACC) (1:1000, 3662) and phospho-ACC (Ser79) (1:1000, 3661), NQO1 (1:500, 62,262), nuclear respiratory factor 1 (NRF1) (1:500, 12,381), nuclear factor-E2-related factor 2 (NRF2) (1:500, 4399), phospho-IRS-1 (Ser307) (1:500, 2381), IκΒα (1:500, 9242), p65 (1:500, 3034), total AMP-activated protein kinase (AMPK) (1:1000, 2532) and phospho-AMPK (Thr172) (1:1000, 2531) (all from Cell Signaling Technology, Danvers, MA, USA; numbers indicate catalogue number), oxidative phosphorylation (1:1000, ab110413) (OXPHOS), peroxisome proliferator-activated receptor γ co-activator 1α (PGC-1α; (1:1000, ab54481) (Abcam), OCT-1 (1:500, sc-8024X), peroxisome proliferator-activated receptor (PPAR)β/δ (1:500, sc-7197), prohibitin (1:500, sc-377037), suppressor of cytokine signalling 3 (SOCS3) (1:500, sc-51699), Tribbles 3 (TRB3) (1:500, sc-365842), glyceraldehyde 3-phosphate dehydrogenase (1:500, sc-32233), total IRS-1 (1:500, sc-560) and β-actin (1:500, sc-47778) (all from Santa Cruz; numbers indicate catalogue number). Detection was achieved using the Western Lightning Plus-ECL chemiluminescence kit (PerkinElmer, Waltham, MA, USA). The equal loading of proteins was assessed by Ponceau S staining. For validation, we used a protein marker (Precision Plus Protein Dual Color Standards 1610374; Bio-Rad, Hercules, CA, USA), on the same blots. All of these commercially available antibodies showed a single distinct band at the molecular weight indicated in the datasheets.
Electrophoretic mobility shift assay
The electrophoretic mobility shift assay (EMSA) was performed as described in ESM Methods.
Glucose uptake experiments were performed as described in ESM Methods.
The chemiluminescent blots were imaged using the ChemiDoc MP imager (Bio-Rad). Image acquisition and subsequent densitometric analysis of the corresponding blots were performed using ImageLab software version 4.1 (Bio-Rad). For further details, see ESM Methods.
Results were normalised to levels in control groups and are expressed as mean ± SD. Significant differences were established by either Student’s t test or two-way ANOVA, according to the number of groups compared, using GraphPad Prism V4.03 software (GraphPad Software, San Diego, CA, USA). When significant variations were found by two-way ANOVA, the Tukey–Kramer multiple comparison post hoc test was performed. Differences were considered significant at p < 0.05.
VLDL induces ER stress, inflammation and insulin resistance in myotubes
When we examined proteins involved in the insulin signalling pathway, we observed that in agreement with a previous study reporting that ER stress reduced insulin receptor levels in adipocytes , protein levels of IRβ were reduced in VLDL-exposed cells (Fig. 2d). In addition, VLDL increased IRS-1 phosphorylation at Ser307 (Fig. 2d) and blunted insulin-stimulated Akt phosphorylation (Fig. 2e).
VLDL increases ER stress, mitochondrial dysfunction and inflammation in isolated skeletal muscle
ERK1/2 inhibition prevents the effects of VLDL
ApoCIII mimics the effects of VLDL through TLR2
Although it is well established that insulin resistance drives atherogenic dyslipidaemia, there is little evidence on whether the increase in VLDL particles associated with insulin-resistant states exacerbates the insulin resistance. Our findings demonstrate that exposure of myotubes and isolated skeletal muscle to VLDL increases the levels of ER stress and inflammatory markers and attenuates the insulin signalling pathway. These data indicate that increased levels of VLDL particles may contribute towards exacerbation of insulin resistance. Our findings also demonstrate that apoCIII may be the VLDL component responsible for the changes caused by VLDL exposure. This is interesting, since apoCIII expression is increased by insulin deficiency, insulin resistance [33, 34] and hyperglycaemia , converting apoCIII into the most abundant VLDL apolipoprotein in individuals with diabetes , suggesting that the increase in apoCIII levels in diabetic states may contribute to exacerbation of these conditions. In this regard, it is interesting to note that humans with a mutation in the APOCIII gene (also known as APOC3) that results in a reduction in the half-life of apoCIII, show a favourable lipoprotein pattern, increased insulin sensitivity and longevity and protection against cardiovascular diseases [36, 37]. Recent evidence seems to confirm that apoCIII plays a key role in diabetes. Thus, decreasing apoCIII in mice results in improved glucose tolerance . In agreement with this, antisense-mediated lowering of plasma apoCIII improves dyslipidaemia and insulin sensitivity in humans with type 2 diabetes  and a null mutation in human APOCIII confers a favourable plasma lipid profile, although it does not improve insulin sensitivity .
The mechanism by which VLDL and apoCIII increase ER stress and inflammation and attenuate insulin signalling in myotubes seems to involve ERK1/2 activation. This kinase has been implicated in the development of insulin resistance associated with obesity and type 2 diabetes . In fact, Erk1 −/− mice (also known as Mapk3 −/− mice) challenged with a high-fat diet are resistant to obesity and are protected from insulin resistance . In addition, hyperinsulinaemic–euglycaemic clamp studies have demonstrated an increase in whole-body insulin sensitivity in ob/ob-Erk1 −/− mice associated with an increase in both insulin-stimulated glucose disposal in skeletal muscles and adipose tissue insulin sensitivity .
In the present study, apoCIII-induced ERK1/2 activation was accompanied by a reduction in AMPK activity. An inhibitory crosstalk exists between AMPK and ERK1/2 and activation of ERK1/2 inhibits AMPK and promotes ER stress-induced insulin resistance in skeletal muscle cells [14, 29]. Hence, VLDL and apoCIII-induced ER stress might be a result of the reduction in AMPK activity. In fact, AMPK activation inhibits ER stress [14, 43], whereas the reduction in its activity promotes ER stress . Moreover, VLDL- and apoCIII-induced ER stress ultimately results in activation of the IKKβ–NF-κΒ pathway, which attenuates the insulin signalling pathway by phosphorylating IRS-1 in serine residues and increases the transcription of inflammatory genes. In agreement with this, we found that ERK1/2 inhibition or knockdown prevented the changes in ER stress and inflammation and the attenuation of the insulin signalling pathway caused by VLDL. Moreover, ERK1/2 inhibition prevented the reduction in AMPK caused by apoCIII, confirming the negative crosstalk between ERK1/2 and AMPK.
Similarly, the reduction in AMPK caused by apoCIII-induced ERK1/2 activation may contribute to reduced PGC-1α levels, since PGC-1α is an important mediator of AMPK-induced gene expression and AMPK activation regulates PGC-1α transcription . Given the key role of PGC-1α in regulating the activity of transcription factors involved in FAO, such as PPARs , the reduction in PGC-1α following treatment with VLDL or apoCIII leads to a decrease in the expression of genes involved in FAO, suggesting that it can promote the deleterious effects of saturated fatty acids .
VLDLs also bind to the VLDL receptor, which is a determinant factor in adipose tissue inflammation and adipocyte macrophage infiltration when stimulated with VLDL from hyperlipidaemic mice . Although we cannot discount a role for this receptor, the fact that the effects of VLDL from normolipidaemic individuals are mimicked by apoCIII seems to suggest that most of the effects of these lipoproteins are caused by the presence of apoCIII in these particles.
Interestingly, our findings indicate that the effects of apoCIII are mediated by TLR2. TLR2 not only recognises numerous lipid-containing molecules but also it recognises endogenous proteins . It is expressed in skeletal muscle cells and is involved in fatty acid-induced insulin resistance . Moreover, activation of the TLR2 pathway ultimately leads to NF-κB and ERK1/2 activation . Likewise, TLR2 deficiency improves insulin sensitivity and attenuates cytokine expression . Our findings confirm the importance of TLR2 in insulin resistance and indicate that its activation by VLDL and apoCIII induces ER stress, inflammation and insulin resistance.
In conclusion, our findings show that VLDL- and apoCIII-induced TLR2 activation results in ER stress, inflammation and insulin resistance by activating ERK1/2 in skeletal muscle cells. These results imply that elevated VLDL in diabetic states can contribute to the exacerbation of insulin resistance.
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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