Endoplasmic reticulum stress does not mediate palmitate-induced insulin resistance in mouse and human muscle cells
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- Hage Hassan, R., Hainault, I., Vilquin, J. et al. Diabetologia (2012) 55: 204. doi:10.1007/s00125-011-2328-9
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Recent experiments in liver and adipocyte cell lines indicate that palmitate can induce endoplasmic reticulum (ER) stress. Since it has been shown that ER stress can interfere with insulin signalling, our hypothesis was that the deleterious action of palmitate on the insulin signalling pathway in muscle cells could also involve ER stress.
We used C2C12 and human myotubes that were treated either with palmitate or tunicamycin. Total lysates and RNA were prepared for western blotting or quantitative RT-PCR respectively. Glycogen synthesis was assessed by [14C]glucose incorporation.
Incubation of myotubes with palmitate or tunicamycin inhibited insulin-stimulated protein kinase B (PKB)/ v-akt murine thymoma viral oncogene homologue 1 (Akt). In parallel, an increase in ER stress markers was observed. Pre-incubation with chemical chaperones that reduce ER stress only prevented tunicamycin but not palmitate-induced insulin resistance. We hypothesised that ER stress activation levels induced by palmitate may not be high enough to induce insulin resistance, in contrast with tunicamycin-induced ER stress. Indeed, tunicamycin induced a robust activation of the inositol-requiring enzyme 1 (IRE-1)/c-JUN NH2-terminal kinase (JNK) pathway, leading to serine phosphorylation of insulin receptor substrate 1 (IRS-1) and a decrease in IRS-1 tyrosine phosphorylation. In contrast, palmitate only induced a very weak activation of the IRE1/JNK pathway, with no IRS1 serine phosphorylation.
These data show that insulin resistance induced by palmitate is not related to ER stress in muscle cells.
KeywordsAktCeramideInsulin signallingLipidMyotubesProtein kinase BTunicamycinUnfolded protein response
v-akt murine thymoma viral oncogene homologue 1
Activating transcription factor 4
Activating transcription factor 6
Binding immunoglobulin protein
CCAAT/enhancer binding protein (C/EBP) homologous protein
Eukaryotic initiation factor 2α
Glycogen synthase kinase
Glucose regulated protein 78
Insulin receptor substrate-1
c-Jun NH2-terminal kinase
4-Phenyl butyric acid
Protein kinase R-like ER protein kinase
Protein kinase B
Protein kinase C ζ
Protein phosphatase 2A
Unfolded protein response
X box binding protein 1
Insulin resistance observed in skeletal muscle is a main feature of obesity and type 2 diabetes. Elevated plasma concentrations of NEFA are consistently observed, leading to lipotoxicity through the over-accumulation of lipid derivatives such as diacylglycerol and ceramides in non-adipose tissues [1, 2]. Palmitate, a predominant saturated NEFA, acts indirectly on insulin signalling by inducing de novo ceramide synthesis and it is now clear that ceramides accumulate in the skeletal muscle of different rodent models of insulin resistance [3, 4], as well as in the muscles of obese insulin-resistant humans [5, 6]. Ceramides modulate diverse signalling pathways through the inhibition of pro-survival kinases such as protein kinase B (PKB)/v-akt murine thymoma viral oncogene homologue 1 (Akt) and the activation of stress-activated kinases such as c-Jun kinase (JNK) and protein kinase C zeta (PKCζ), as well as protein phosphatases such as protein phosphatase 2A (PP2A) and protein phosphatase 1 [7–9]. We, and others, have shown that palmitate-generated ceramides inhibit insulin signalling in muscle cells, specifically by targeting PKB/Akt [10–12], a key serine/threonine kinase implicated in the stimulation of glycogen synthesis and glucose transport by insulin in muscle . Several studies in which ceramide biosynthesis was prevented pharmacologically or genetically have demonstrated that accumulation of ceramide inhibits insulin signalling and, therefore, induced insulin resistance [10, 14, 15].
Interesting observations indicate that saturated fatty acids may also act at the level of the endoplasmic reticulum (ER) by modulating the fusion/fission events of membranes [16, 17]. The result is a dilatation of the ER and the accumulation of unfolded or misfolded protein within the ER lumen. To deal with this stress, cells induce a response called unfolded protein response (UPR) . Three main effectors are activated to restore ER function: protein kinase R-like ER protein kinase (PERK), activating transcription factor 6 (ATF6) and inositol-requiring enzyme-1 (IRE-1) . In the basal state, these factors are kept inactive through their association with a protein chaperone called binding immunoglobulin protein (BIP)/glucose regulated protein 78 (GRP78) in the ER. Following ER stress, BIP/GRP78 is displaced and interacts with unfolded proteins , allowing the activation of both PERK and IRE1 by trans-autophosphorylation, and ATF6 by proteolytic cleavage . Once activated these pathways induce several genes and chaperones, such as activating transcription factor 4 (ATF4), CCAAT/enhancer binding protein [C/EBP] homologous protein (CHOP) and BIP/GRP-78, to restore ER homeostasis. The activation of PERK leads to the phosphorylation of eukaryotic initiation factor 2α (eiF2α) resulting in global attenuation of protein synthesis . IRE-1 has an endonuclease activity that activates the transcription factor X box binding protein 1 (XBP-1) by removing a 26 bp segment of its mRNA . Ultimately, when ER homeostasis cannot be restored, apoptosis is initiated .
Several studies carried out in liver and adipose tissue have shown that nutrient excess-induced chronic ER stress plays a role in the pathogenesis of diabetes, contributing to hepatic steatosis , beta cell loss and insulin resistance [21–23]. Insulin receptor substrate 1 (IRS-1), a docking protein downstream of the insulin receptor , is a target of prolonged ER stress through its inhibition by the IRE-1/c-Jun NH2-terminal kinase (JNK) pathway . Reciprocally, prevention of ER stress by the use of chemical chaperones improves glucose homeostasis and insulin sensitivity [25–27].
Activation of ER stress markers in skeletal muscle is still contradictory. ER stress has also been shown to take place in mouse skeletal muscle in response to palmitate , and recent studies have shown that UPR takes place in skeletal muscles of diabetic patients  and in those of mice fed a high-fat diet . On the other hand, other studies failed to detect ER stress in the skeletal muscles of obese mice , as well as of obese  and insulin-resistant  patients.
Because ceramide biosynthesis from palmitate occurs at the level of the ER , and since the activation of the ER can reciprocally induce ceramide biosynthesis in INS-1 cells , we questioned the relative roles of ER stress and ceramides in the deleterious action of palmitate on the insulin signalling pathway in muscle cells. We demonstrate that palmitate induces an ER stress in muscle cells, inhibits the insulin signalling pathway and alters glucose metabolism through the production of ceramides. However, inhibition of the palmitate-induced ER stress did not improve palmitate-induced insulin resistance in muscle cells, suggesting that the negative action of palmitate on insulin signalling through ceramide production is independent of ER stress induction.
All reagent-grade chemicals, insulin, palmitate, 4-phenyl butyric acid (PBA), and BSA were purchased from Sigma-Aldrich (St Louis, MO, USA). C2-ceramide and tauroursodeoxycholic acid (TUDCA) were obtained from Calbiochem (Darmstadt, Germany). Complete protein phosphatase inhibitor tablets were obtained from Boehringer-Roche Diagnostics (Mannheim, Germany). Antibodies against native PKB/Akt, 473Ser PKB/Akt, 183Thr /185Tyr JNK, β-actin, 980Thr PERK, 21/9Ser glycogen synthase kinase (GSK3)α/β and 51Ser eiF2α were from Cell Signaling (New England Biolabs, Boston, MA, USA), GRP-78 from Santa Cruz Biotechnology (Heidelberg, Germany), 724Ser IRE-1 from Novus Biologicals (Littleton, CO, USA), and 307Ser IRS-1 and 612Tyr IRS-1 from Upstate (Lake Placid, NY, USA). Horseradish peroxidase anti-rabbit, -mouse and -sheep/goat IgGs were from Jackson ImmunoResearch Laboratories (West Grove, PA, USA) and the enhanced chemiluminescent substrate was from Thermo Scientific Pierce (Rockford, IL, USA). D-[U-14C]glucose was obtained from PerkinElmer (Courtaboeuf, France).
C2C12 myoblasts were maintained at 37°C in DMEM (Invitrogen, Paisley, UK) containing 10% (wt/vol.) fetal bovine serum. For differentiation into myotubes, the myoblasts were grown to confluency and the medium was replaced with DMEM containing 2% (wt/vol.) horse serum. Myotubes were used for experiments 4 days following differentiation.
Human skeletal muscle cells
Muscle biopsies were obtained in the context of approved preclinical and clinical trials , and via the Tissue Bank for Research (Myobank) of the French Association against Myopathies (AFM), in agreement with the French bioethical law (law no 94–654 of 29 July 1994, modified 22 January 2002) on informed consent. Donors were adults and had no clinical signs of muscular disease. Fresh muscle biopsies were minced and enzymatically dissociated with collagenase. Cell myoblasts were purified, grown and differentiated as myotubes as previously described .
Preparation of whole cell lysates
Cells were lysed following experimental manipulation (see figure legends) in an appropriate volume of lysis buffer .
Cell lysates were subjected to SDS/PAGE and immunoblotted as previously reported . Nitrocellulose membranes were probed with various antibodies as described in the figure legends. Detection of primary antibodies was performed using appropriate peroxidase-conjugated IgGs and protein signals were visualised using enhanced chemiluminescence (Thermo Scientific Pierce) by exposure to Kodak autoradiographic film.
Fatty acid treatment
Cells were treated with palmitate conjugated with fatty acid free BSA as described . Briefly, fatty acids were dissolved in ethanol and diluted 1:10 in DMEM containing 10% (wt/vol.) BSA. Final BSA concentration in the culture was 1.5%. Concentrations of palmitate are indicated in the figure legends.
C2C12 myotubes were pre-incubated with 0.75 mmol/l palmitate with or without 1 mmol/l TUDCA for 16 h. Five hours before the experiment, cells were deprived of serum in DMEM containing palmitate with or without TUDCA. Cells were then washed with PBS and incubated with or without 100 nmol/l insulin for 60 min in PBS containing D-[U-14C] glucose (3.7 × 10−3 MBq/ml). The incubation was terminated by three washes with ice-cold 0.9% (wt/vol.) NaCl prior to lysis in 60% (wt/vol.) KOH. Cellular glycogen was precipitated from lysates as described previously , and associated radioactivity was determined by liquid scintillation counting. Protein was determined using the Bradford method .
Isolation of total RNA and quantitative RT-PCR
Total RNA was isolated as described previously . We retro-transcribed 1 μg RNA using Superscript II (Invitrogen). Real-time quantitative RT-PCR (qRT-PCR) analyses were performed with 25 ng cDNA and 250 nmol/l sense and antisense primers (Eurogentec, Angers, France) in a final reaction volume of 25 μl by using the qPCR Core Kit (Eurogentec) and the MyiQ real-time PCR detection system (Bio-Rad). Specific primers were designed using Primer Express software (version 1.0; Applied Biosystems, Carlsbad, CA, USA). Relative quantification of each gene was calculated after normalisation to 18S ribosomal RNA by using the comparative Ct method. For analysis of Xbp1 mRNA splicing, 200 ng cDNA was amplified with specific primers for the rat Xbp1 gene (forward, 5′-CCATGGGAAGATGTTCTGGG-3′; reverse, 5′-ACACGCTTGGGGATGAATGC-3′). The conditions used for the PCR have been described previously . PCR products were separated by electrophoresis on 2.5% agarose gel and visualised by ethidium bromide staining.
Results are expressed as mean ± SEM. Statistical significance was assessed using one-way ANOVA followed by a Bartlett test. A p value <0.05 was considered significant.
Palmitate activates ER stress and alters insulin signalling in C2C12 myotubes
Interestingly, palmitate also triggered a UPR in C2C12 myotubes (Fig. 1). An increase in ER stress markers was observed in response to palmitate, i.e. phosphorylation of the translation initiation factor eIF2α (Fig. 1 a,b) splicing of Xbp1 mRNA (Fig. 1c), and activation by two- to fourfold of several ER stress chaperones and foldases, such as the protein content of GRP78 (Fig. 1a, b), and Chop (also known as Ddit3), Atf4, oxygen regulated protein 150 (Orp150 [also known as Hyou1]) and Trb3 (also known as Trib3) mRNA (Fig. 3b).
Ceramides mediate the action of palmitate on both PKB/Akt and the UPR
ER stress does not mediate palmitate-induced alterations in insulin signalling in C2C12 and human muscle cells
We repeated the experiments on insulin signalling in C2C12 cells using another ER stress inhibitor, namely 4-phenylbutyric acid (PBA) . As shown with TUDCA, PBA reversed palmitate-induced ER stress but insulin signalling was not improved (Fig. 3c, d).
In order to increase the physiological relevance of these findings, we repeated these experiments in cultured human myotubes. In human myotubes, palmitate induces an ER stress (Fig. 3e) and impairs insulin signalling. As is the case in rodent cells, TUDCA, despite alleviating the ER stress, did not restore insulin signalling (Fig. 3e, f).
Tunicamycin-induced ER stress alters insulin signalling in C2C12 myotubes
Tunicamycin activates the IRE-1/JNK pathway to a greater extent than palmitate
Considering the events downstream of IRE-1 activation, the level of Xbp1 splicing reflected the higher phosphorylation of IRE-1 by tunicamycin, compared with what was observed with palmitate (Fig. 6b) and phosphorylation of JNK was more prominent in cells treated with tunicamycin than with palmitate (Fig. 6c). It has been shown in liver cells that the insulin signalling pathway was affected after tunicamycin treatment through the inhibition of the docking protein IRS-1 when phosphorylated on serine residues by JNK . Tunicamycin-treated C2C12 myotubes displayed a significant increase in phosphorylation of IRS-1 on serine 307 and a drastic decrease in insulin-induced IRS-1 tyrosine phosphorylation (Fig. 6d). In contrast, and as already described , palmitate-treated C2C12 cells did not exhibit any serine phosphorylation of IRS-1 (Fig. 6d) and IRS-1 tyrosine phosphorylation was not affected. This confirms the lack of activation of the IRE-1/JNK pathway by palmitate in these cells and corroborates a more downstream action of palmitate on the insulin signalling pathway at the level of PKB/Akt.
Palmitate induces concomitantly an ER stress and insulin resistance in muscle cells
Accumulation of unfolded proteins in the ER lumen induces an adaptive response called UPR in order to decrease protein synthesis and to restore ER folding capacities . However, in situations where ER stress is activated chronically, a decline of insulin action appears in liver and adipose tissue, thus contributing to the development of type 2 diabetes . Although a direct role of ER stress in the pathogenesis of diabetes and obesity has been demonstrated in both liver and adipose cells [19, 20, 22, 23, 25], much less information is available in skeletal muscle. Studies have shown the appearance of ER stress markers in response to high-fat diet in mouse skeletal muscles  or in diabetic patients , but very recently, the involvement of ER stress in human skeletal muscle at the onset of glucose intolerance has been questioned [30, 31]. In the present study, we examined the direct effects of NEFA (i.e. palmitate) on both muscle cell ER homeostasis and insulin sensitivity. Our data demonstrate that palmitate concomitantly disrupts ER homeostasis and alters insulin signalling and glucose metabolism, but that palmitate-induced ER stress is not responsible for the decrease in insulin sensitivity.
Palmitate-induced ER stress is linked to ceramide production
Interestingly, and in contrast with what had been shown in INS-1 cells  and H4IIE liver cells , de novo ceramide synthesis from palmitate was required for fatty acid-induced ER stress in C2C12 myotubes. Indeed, we show that ceramides alone can induce an ER stress and that inhibition of ceramide synthesis in the presence of palmitate prevents ER stress in muscle cells. The action of ceramides on ER stress has also been described in various cell types such as prostate epithelial cells , mouse T hybridoma cells , pancreatic beta cells  and human glioma cells . However, the mechanism by which ceramides can induce an ER stress remains largely unknown.
Tunicamycin-induced ER stress promotes an insulin resistance in muscle cells
As the activation of ER stress itself can be involved in the development of insulin resistance, we have reproduced a situation of high ER stress without any added NEFA that could be a substrate for ceramide production. Our data indicate that activation of ER stress by tunicamycin downregulates the insulin signalling pathway in C2C12 myotubes, an effect which was reversed in the presence of a chemical chaperone that reduced the intensity of ER stress. Tunicamycin strongly activates the IRE-1/JNK pathway, leading to serine phosphorylation of IRS-1 and a blockade of insulin signalling at this step. Insulin resistance induced by tunicamycin occurred independently of any ceramide production, since tunicamycin action was not prevented when cells were pre-incubated with myriocin (data not shown).
Palmitate-induced insulin resistance is not linked to the concomitant palmitate-induced ER stress in muscle cells
Surprisingly, and despite the fact that a tunicamycin-induced ER stress was able to inhibit insulin signalling (see above), ER stress induced by palmitate does not play a role in the induction of insulin resistance. Indeed, lowering the ER stress with two chemical chaperones (TUDCA and PBA) did not improve insulin sensitivity or glucose metabolism in rodent and human muscle cells, in contrast with what was observed when ER stress was induced by tunicamycin.
ER stress induced by palmitate is not strong enough to induce insulin resistance
We then tried to understand why palmitate-induced ER stress does not play a role in the initiation of insulin resistance in muscle cells. One potential answer was that palmitate could modulate only selective genes of the UPR and/or that palmitate did not induce ER stress to a sufficiently high level to activate the whole UPR system. We confirmed both of these assumptions. We showed that the magnitude of activation of ER stress genes was much higher in cells treated with tunicamycin than with palmitate (compare Figs 3, 5). Importantly, a 50% decrease in tunicamycin-induced stress markers in the presence of TUDCA is sufficient to prevent the ER stress-mediated tunicamycin action on insulin signalling. This demonstrates that ER stress activation has to be strong to play a deleterious action on insulin signalling. In particular, the IRE-1/JNK pathway was poorly activated after palmitate treatment, but fully activated with tunicamycin. The modest activation of this pathway by palmitate (or directly with ceramides) explains why, in contrast with tunicamycin, palmitate and ceramides have no negative impact on IRS-1 in muscle cells and act only at the level of PKB/Akt, as described previously [8, 10, 12]. A difference in ER stress activation between palmitate and chemical agents such as tunicamycin had already been observed in H4IIE liver cells . Indeed, in that study, palmitate induced the UPR only moderately, with no real action on the IRE-1/XBP-1 pathway, compared with what was observed in cells treated with tunicamycin .
We must point out that the cellular models used in this study to investigate an individual fatty acid remains far from an in vivo situation where a mixture of fatty acids is always chronically present. In previous studies, it has been shown that unsaturated fatty acids such as oleate could prevent both palmitate-induced ER stress-mediated apoptosis and insulin resistance in liver and muscle cells by diverting palmitate from producing ceramide to neutral lipid storage [43, 44]. Thus, the balance between saturated and unsaturated fatty acids remains very important for the study of their impact on glucose metabolism.
During the course of our study, an article has challenged our results. Peng et al. have shown that palmitate-induced insulin resistance was mediated by ER stress in C2C12 cells . It is hard to explain the discrepancy between this study and our data, but potential explanations could be first, that they were using undifferentiated C2C12 myoblasts for their study and second, that added palmitate was not complexed to albumin leading to supraphysiological free palmitate concentrations.
We are grateful to P. Guicheney for helpful advice, to X. Le Liepvre for technical help and to the tissue bank of the Association Française des Myopathies for human biopsy samples. R. Hage Hassan was supported by the French Ministry of Research.
All authors were responsible for the conception and design of the study, analysis and interpretation of the data, drafting and revising the manuscript. All authors approved the final version to be published.
Duality of interest
All authors declare that there is no duality of interest associated with this manuscript.