Dilinoleoyl-phosphatidic acid mediates reduced IRS-1 tyrosine phosphorylation in rat skeletal muscle cells and mouse muscle
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Insulin resistance in skeletal muscle is strongly associated with lipid oversupply, but the intracellular metabolites and underlying mechanisms are unclear. We therefore sought to identify the lipid intermediates through which the common unsaturated fatty acid linoleate causes defects in IRS-1 signalling in L6 myotubes and mouse skeletal muscle.
Materials and methods
Cells were pre-treated with 1 mmol/l linoleate for 24 h. Subsequent insulin-stimulated IRS-1 tyrosine phosphorylation and its association with the p85 subunit of phosphatidylinositol 3-kinase were determined by immunoblotting. Intracellular lipid species and protein kinase C activation were modulated by overexpression of diacylglycerol kinase ɛ, which preferentially converts unsaturated diacylglycerol into phosphatidic acid, or by inhibition of lysophosphatidic acid acyl transferase with lisofylline, which reduces phosphatidic acid synthesis. Phosphatidic acid species in linoleate-treated cells or muscle from insulin-resistant mice fed a safflower oil-based high-fat diet that was rich in linoleate were analysed by mass spectrometry.
Linoleate pretreatment reduced IRS-1 tyrosine phosphorylation and p85 association. Overexpression of diacylglycerol kinase ɛ reversed the activation of protein kinase C isoforms by linoleate, but paradoxically further diminished IRS-1 tyrosine phosphorylation. Conversely, lisofylline treatment restored IRS-1 phosphorylation. Mass spectrometry indicated that the dilinoleoyl-phosphatidic acid content increased from undetectable levels to almost 20% of total phosphatidic acid in L6 cells and to 8% of total in the muscle of mice fed a high-fat diet. Micelles containing dilinoleoyl-phosphatidic acid specifically inhibited IRS-1 tyrosine phosphorylation and glycogen synthesis in L6 cells.
These data indicate that linoleate-derived phosphatidic acid is a novel lipid species that contributes independently of protein kinase C to IRS-1 signalling defects in muscle cells in response to lipid oversupply.
KeywordsDiacylglycerol Fatty acid Insulin resistance IRS-1 L6 muscle cells Phosphatidic acid Protein kinase C Skeletal muscle
green fluorescent protein
intraperitoneal glucose tolerance test
lysophosphatidic acid acyltransferase
novel protein kinase C
peroxisome proliferator-activated receptor
protein kinase B
protein kinase C
Insulin resistance is a reduced capacity of peripheral tissues to respond to physiological levels of circulating insulin and is recognised as a major contributory factor to the development of type 2 diabetes. Insulin resistance in skeletal muscle is particularly significant because of the major role of this tissue in glucose homeostasis ; it is strongly correlated with lipid oversupply . The mechanisms by which lipids can generate insulin resistance are therefore an important focus for investigation.
The metabolic and mitogenic actions of insulin are initiated when the hormone binds to its receptor and stimulates a signalling cascade by activation of receptor tyrosine kinase activity and tyrosine phosphorylation of IRSs such as IRS-1 . In contrast, many agents that induce insulin resistance cause phosphorylation of IRS-1 on serine residues, leading to the dissociation of IRS-1 from the insulin receptor, reduced tyrosine phosphorylation and phosphatidylinositol 3-kinase (PI3K) association, and ultimately IRS-1 degradation [4, 5].
Pathophysiological levels of lipid availability have been associated with reduced IRS-1 tyrosine phosphorylation and associated PI3K activity in muscle [6, 7]. It therefore seems likely that the activation of specific lipid-activated signalling pathways that interfere with normal insulin action play a part in the generation of insulin resistance. A link between isoforms of the lipid-activated protein kinase C (PKC) family and insulin resistance has been reported in several studies [8, 9, 10, 11, 12].
While the signalling pathways initiated by NEFA oversupply that are involved in the generation of insulin resistance have been partly elucidated [6, 7], less is known about which intermediate lipid species are directly involved. One candidate is the lipid second messenger diacylglycerol (DAG), a potent activator of conventional and novel PKC (nPKC) isoforms, which can be elevated through de novo synthesis from NEFAs. Elevated DAG levels have been demonstrated in rodent and human models of insulin resistance associated with increased lipid availability [8, 10, 13]. A second mechanism, specifically relating to the manner in which saturated lipids can cause insulin resistance, involves generation of the lipid intermediate ceramide [14, 15, 16]. Thus different lipid species can interfere with insulin signalling by different means. However, while DAG or ceramide formation is unlikely to account for all lipid-mediated insulin resistance, little is known about other NEFA-derived inhibitors of insulin action. Here, we show that the unsaturated NEFA linoleate, a key NEFA found in diets, causes a reduction in IRS-1 tyrosine phosphorylation in myotubes. While we found no evidence of a role for DAG in this model, we instead identified a novel role for the NEFA-derived second messenger phosphatidic acid (PtdH), in particular dilinoleoyl-PtdH.
Materials and methods
Cell culture, fatty acid pre-treatment and insulin stimulation
L6 rat skeletal muscle cells were a kind gift from A. Klip (Hospital for Sick Children, Toronto, ON, Canada). Cells were maintained, differentiated and pre-treated with 1 mmol/l linoleate for 24 h as previously described , with the exception that cells were serum-starved for the final 4 h of treatment. Cells were stimulated with 100 nmol/l insulin for 10 min. Glycogen synthesis was measured as previously .
Generation and use of recombinant adenovirus and immunoblot analyses
Lipid determination by thin layer chromatography and mass spectrometry
Cellular DAG was measured by a method adapted from that described previously by Nakamura and Handa . For the investigation of lipid compartmentalisation, cell homogenates were subjected to sucrose gradient flotation [20, 21]. Phospholipids were separated by two-dimensional thin-layer chromatography (TLC) . Electrospray ionisation mass spectrometry was performed on a Waters Quattro Micro equipped with an electrospray ion source and controlled by Micromass Masslynx version 4.0 software (Waters, Manchester, UK). Detailed methods are given in the ESM.
Comparison of phosphatidic acid species in muscle from mice fed chow or a high-fat diet
Glucose intolerance in mice fed a high-fat diet  was confirmed by intraperitoneal glucose tolerance test (ipGTT) . Lipid extracts of quadriceps muscles were subjected to mass spectrometry. For further details, see ESM.
Phosphatidic acid treatment
PtdH-containing micelles were generated using either dipalmitoyl-PtdH (16:0/16:0), palmitoyl-oleoyl-PtdH (16:0/18:1), palmitoyl-linoleoyl-PtdH (16:0/18:2) or dilinoleoyl-PtdH (18:2/18:2) (Avanti, Alabaster, AL, USA), together with a 4:1 excess of phosphatidylcholine (Sigma Chemical, St Louis, MO, USA). Lipids in 2:1 (vol./vol.) chloroform:methanol were mixed, dried under nitrogen and sonicated into 500 μl serum-free medium. This suspension was diluted 25-fold into minimum essential medium α (containing 2% fetal calf serum [vol./vol.] or serum-free) to give a PtdH concentration of 100 μmol/l. L6 myotubes were treated with PtdH-containing medium for 16 h in the presence of fetal calf serum, followed by 4 h in the absence of serum. Cells were stimulated with insulin and harvested as above.
Results were analysed by Student’s t test or ANOVA using Statview 4.5 for Macintosh (available from http://www.statview.com, last accessed in May 2007). Results are expressed as means±standard error, with differences considered statistically significant at p < 0.05.
Linoleate pre-treatment of L6 myotubes reduces insulin-stimulated IRS-1 tyrosine phosphorylation
Evidence that PtdH, rather than DAG, mediates linoleate-induced IRS-1 depletion
To determine the importance of such DAG species in our model, we examined the effects of diacylglycerol kinase (DAGK)ɛ overexpression. This isoform of DAGK preferentially catalyses the conversion of DAG comprising polyunsaturated acyl chains to PtdH, and hence promotes the termination of PKC activation . While endogenous expression of DAGKɛ in skeletal muscle has not been reported, we employed the enzyme as a means to diminish signalling species of DAG and to suppress PKC function. L6 cells overexpressing DAGKɛ or green fluorescent protein (GFP) as control were pre-treated with linoleate. In control cells this caused chronic activation of nPKC isoforms. This was evidenced by translocation of nPKCɛ from a cytosolic to a membrane fraction and by the downregulation of cytosolic nPKCδ, also indicative of chronic activation (Fig. 2e). While nPKCθ has been implicated in muscle insulin resistance caused by lipid infusion , it is poorly expressed in myotubes [18, 27] and its overexpression does not inhibit insulin action in these cells . This isoform was therefore not addressed in the present study. Linoleate had minimal effects on the conventional PKC isoforms, PKCα and PKCβ (Fig. 2e). Importantly, DAGKɛ overexpression inhibited the chronic activation of nPKCɛ by linoleate, as indicated by its cytosolic localisation, and also reduced the downregulation of nPKCδ (Fig. 2e). These results demonstrate that DAGKɛ overexpression is a useful tool for preventing the chronic activation of nPKC isoforms in myotubes by lipid pre-treatment.
To address this unexpected possibility, we employed lisofylline, an inhibitor of lysophosphatidic acid acyltransferase (LPAAT). LPAAT catalyses the formation of PtdH from lyso-PtdH and acyl-CoA, so its inhibition would be expected to reduce PtdH formation from linoleate. The presence of lisofylline had reciprocal effects compared with DAGKɛ overexpression, in that the inhibitor restored insulin-stimulated IRS-1 tyrosine phosphorylation in linoleate-pretreated myotubes and also enhanced total IRS-1 levels (Fig. 3d–f). These results are again consistent with a role for PtdH in the defective IRS-1 tyrosine phosphorylation induced by linoleate.
Linoleate treatment increases turnover of cellular PtdH
Linoleate induces the appearance of specific PtdH species
Relative abundance of PtdH molecular species in L6 myotubes pretreated in the absence or presence of linoleate
6 h, control (%)
6 h, linoleate (%)
24 h, control (%)
24 h, linoleate (%)
Elevation of 18:2/18:2-PtdH in skeletal muscle from mice fed a high-fat diet
Phosphatidic acid composition of skeletal muscle from mice fed chow or a high-fat diet
Chow (nmol/g tissue)
High-fat diet (nmol/g tissue)
13.9 ± 1.4
6.2 ± 1.3*
12.3 ± 2.1
22.8 ± 1.9*
21.6 ± 1.5
9.1 ± 2.1**
15.4 ± 1.3
12.3 ± 1.6
0.0 ± 0.0
8.3 ± 0.8***
8.4 ± 2.0
8.5 ± 1.0
12.2 ± 1.6
11.5 ± 1.3
15.8 ± 2.3
4.0 ± 1.3**
41.4 ± 0.4
21.0 ± 2.6***
Specific inhibition of insulin-stimulated IRS-1 tyrosine phosphorylation by 18:2/18:2-PtdH
We have previously shown that linoleate inhibits glycogen synthesis in cultured myotubes [14, 17]. To determine whether 18:2/18:2-PtdH could also specifically mimic this aspect of linoleate-induced insulin resistance, we measured glycogen synthesis after cells had been pretreated in the absence or presence of PtdH-containing micelles. While micelles themselves reduced insulin-stimulated glycogen synthesis in the absence of any PtdH, only 18:2/18:2-PtdH was able to inhibit this further, by approximately 50% (Fig. 6d). The effects of 18:2/18:2-PtdH on IRS-1, as well as the demonstration of the inhibition of a metabolic endpoint of insulin action by the lipid, further support an inhibitory effect of linoleate-derived PtdH on insulin action and indicate a level of specificity dictated by the acyl side chains.
We show here that the unsaturated NEFA linoleate induces insulin resistance in L6 myotubes at the level of IRS-1 tyrosine phosphorylation and its subsequent association with PI3K. In addition, we demonstrated that linoleate affects the intracellular PtdH profile and that direct modulation of PtdH can also affect IRS-1 phosphorylation. Further, our results are consistent with an inhibitory role for 18:2/18:2-PtdH, rather than for DAG and nPKCs.
The absence of a change in total DAG effected by linoleate pre-treatment, despite elevation of TG, is counter-intuitive, but consistent with previous work [31, 32]. This and the fact that we were unable to show elevation of DAG in a specific compartment by subcellular fractionation suggests that the activation of nPKC isoforms is therefore likely to occur in response to elevation of minor polyunsaturated DAG species. Given the significant increase in TG content in linoleate-pretreated myotubes, the observed alterations in DAG and PtdH species upon lipid treatment probably reflect the role of these lipids as intermediates in de novo synthesis of TG, rather than the activity of phospholipases, which can elevate DAG and PtdH acutely in a signalling context. Nevertheless, linoleate-induced changes in DAG were probably responsible for PKC activation in L6 cells because overexpression of DAGKɛ caused a reversal of the effects of linoleate on nPKC isoforms. Importantly, however, this was unable to prevent the inhibitory effect of linoleate on IRS-1 tyrosine phosphorylation. The lack of evidence that DAG plays a role in the insulin resistance seen in this model is in agreement with our previous findings, made using pharmacological PKC inhibitors and dominant-negative PKC mutants .
The effects of DAGKɛ overexpression and lisofylline pretreatment on IRS-1 tyrosine phosphorylation, taken together with the observations that PtdH turnover was increased in linoleate-treated myotubes, strongly support a role for PtdH itself in the generation of insulin resistance by unsaturated NEFAs. This notion was supported by mass spectrometry data, which indicated that L6 cells treated with linoleate favoured the incorporation of unsaturated 18:2 side chains into PtdH, reflecting their increased availability. This induced the appearance of species such as 18:2/18:2-PtdH, which were undetected in control L6 cells. Our data from mouse skeletal muscle indicated that certain of these species are in fact normally present in intact skeletal muscle, perhaps due to the presence of linoleate in standard chow. However, their levels were significantly elevated in mice fed a high-fat diet, again probably due to increased linoleate availability. A notable exception was the appearance of the novel species 18:2/18:2-PtdH, which had been undetectable in control muscle as well as in untreated L6 cells. The demonstration that only this particular PtdH species was able to inhibit insulin-stimulated IRS-1 tyrosine phosphorylation in L6 myotubes suggests that it may be an important mediator of insulin resistance.
Our experiments involving the use of DAGKɛ, lisofylline or PtdH-containing micelles indicated that IRS-1 protein levels as well as tyrosine phosphorylation could be affected by alterations in PtdH availability. However, changes in tyrosine phosphorylation did not always correspond with altered IRS-1 expression. For example, lisofylline increased IRS-1 levels in both control and linoleate-treated cells, but only restored phosphorylation in the presence of linoleate, rather than increasing it under all conditions. Furthermore, both 16:0/18:2- and 18:2/18:2-PtdH micelles reduced IRS-1 levels, but only 18:2/18:2-PtdH affected tyrosine phosphorylation. These findings suggest that the two effects are at least partly independent and a PtdH-induced mechanism affecting tyrosine phosphorylation, but not IRS-1 levels, may contribute to the diminished IRS-1 phosphorylation in linoleate-treated L6 cells and skeletal muscle . A second mechanism affecting IRS-1 protein levels may additionally occur under harsher experimental conditions. Interestingly, L6 myotubes treated for prolonged periods with linoleate exhibit reduced IRS-1 levels (not shown), while morbidly obese patients have reduced IRS-1 abundance in skeletal muscle , suggesting that such a mechanism may become relevant upon greater lipid accumulation. An additional novel finding of the current study is the increase of IRS-1 protein levels effected by lisofylline. Although currently under evaluation for the treatment of autoimmunity and type 1 diabetes , the potential benefits of this drug for the treatment of insulin resistance and type 2 diabetes have not previously been reported.
Our previous investigations of lipid-treated myotubes demonstrated that unsaturated NEFAs such as oleate and linoleate can inhibit insulin signalling at the level of IRS-1/PI3K, but have no effect on PKB and glycogen synthase kinase 3 [14, 17]. Because these lipids are also able to inhibit insulin-stimulated glucose disposal in this model [14, 17], this may involve a PKB-independent link between IRS-1 and metabolic endpoints. Importantly, 18:2/18:2-PtdH was also able inhibit insulin-stimulated glycogen synthesis in the current study, consistent with a role as a mediator of inhibition of insulin action by linoleate. While oleate is less potent than linoleate , the similarities between the effects of these lipids on myotubes suggest that oleate might also lead to accumulation of inhibitory PtdH species. Because oleate availability can lead to sparing of linoleate β-oxidation and hence accumulation of linoleate-derived glycerolipids , it is conceivable that oleate could cause 18:2/18:2-PtdH accumulation, although this remains to be determined.
Although our models favour accumulation of linoleate, this lipid may still significantly affect glucose homeostasis upon increased availability of mixed lipids in a physiological setting. Compared with a hunter–gatherer diet, linoleate intake has increased approximately fourfold in the Western diet . The insulin resistance caused by saturated and unsaturated NEFAs appears to be mediated by different lipid species (ceramide and PtdH respectively), which act at different sites of insulin signalling (PKB and IRS-1). This may result in the potentiation of inhibitory effects and a greater inhibition of glucose disposal.
While the mechanistic link between PtdH and the inhibition of IRS-1 tyrosine phosphorylation has not been investigated here, we speculate that activation of signalling proteins by the phospholipid may play a role . For example, PtdH has been identified as an activator of mammalian target of rapamycin (mTOR)  and Raf-1 kinase , each of which are upstream of kinase cascades leading to IRS-1 serine phosphorylation [40, 41]. Alternatively, since PtdH is a key lipid involved in membrane biosynthesis and itself modulates membrane properties , the appearance of 18:2/18:2-PtdH may have indirect effects on signalling through changes in cell membrane composition. PtdH could also act at the level of gene expression . Although linoleate itself is a peroxisome proliferator-activated receptor (PPAR)γ agonist  and could promote the expression of enzymes involved in lipid synthesis to influence the availability of inhibitory intermediates, no evidence was observed for linoleate-induced upregulation of PPARγ-dependent genes upon analysis of gene expression in L6 myotubes by transcript profiling using gene arrays or by RT-PCR (Cooney and Breivik, unpublished results). This suggests that PPARγ does not play a significant role in this model of insulin resistance.
In conclusion, we have shown that linoleate reduces IRS-1 tyrosine phosphorylation in L6 cells, which appears to be independent of DAG and nPKC function. Instead, we have indicated a role for PtdH and demonstrated that 18:2/18:2-PtdH, a novel species that is induced in linoleate-treated cells and in insulin-resistant muscle from mice on a high-fat diet, is selectively capable of inhibiting IRS-1 tyrosine phosphorylation. This mechanism may act in concert with others such as the ceramide-mediated inhibition of PKB induced by saturated NEFAs, depending on the NEFA profile of the diet. Finally, we have indicated inhibition of PtdH synthesis by LPAAT as a possible strategy for the treatment of lipid-induced insulin resistance in skeletal muscle.
This work was funded by grants from the National Health and Medical Research Council (NH&MRC) of Australia (to C. Schmitz-Peiffer and T. J. Biden) and Eli Lilly Australia (to C. Schmitz-Peiffer). R. Cazzolli was a recipient of an NH&MRC Dora Lush Postgraduate Research Scholarship. N. Turner is supported by a NH&MRC Peter Doherty Fellowship.
Duality of interest
The authors declare that there is no duality of interest for this work.