Enhanced insulin sensitivity associated with provision of mono and polyunsaturated fatty acids in skeletal muscle cells involves counter modulation of PP2A

International audienceAims/Hypothesis: Reduced skeletal muscle insulin sensitivity is a feature associated with sustained exposure to excess saturated fatty acids (SFA), whereas mono and polyunsaturated fatty acids (MUFA and PUFA) not only improve insulin sensitivity but blunt SFA-induced insulin resistance. The mechanisms by which MUFAs and PUFAs institute these favourable changes remain unclear, but may involve stimulating insulin signalling by counter-modulation/repression of protein phosphatase 2A (PP2A). This study investigated the effects of oleic acid (OA; a MUFA), linoleic acid (LOA; a PUFA) and palmitate (PA; a SFA) in cultured myotubes and determined whether changes in insulin signalling can be attributed to PP2A regulation. Principal Findings: We treated cultured skeletal myotubes with unsaturated and saturated fatty acids and evaluated insulin signalling, phosphorylation and methylation status of the catalytic subunit of PP2A. Unlike PA, sustained incubation of rat or human myotubes with OA or LOA significantly enhanced Akt-and ERK1/2-directed insulin signalling. This was not due to heightened upstream IRS1 or PI3K signalling nor to changes in expression of proteins involved in proximal insulin signalling, but was associated with reduced dephosphorylation/inactivation of Akt and ERK1/2. Consistent with this, PA reduced PP2Ac demethylation and tyrosine 307 phosphorylation-events associated with PP2A activation. In contrast, OA and LOA strongly opposed these PA-induced changes in PP2Ac thus exerting a repressive effect on PP2A.Conclusions/Interpretation: Beneficial gains in insulin sensitivity and the ability of unsaturated fatty acids to oppose palmitate-induced insulin resistance in muscle cells may partly be accounted for by counter-modulation of PP2A

Defect of insulin signal in peripheral tissues: Important role of ceramide

International audienc

Oleic acid (OA) and linoleic acid (LOA) enhance and prolong insulin-dependent phosphorylation of Akt and ERK in L6 myotubes.

<p>The experimental approach used to test the effect of OA and LOA provision on Akt and ERK1/2 phosphorylation is depicted in (A). L6 myotubes were incubated with serum-free media containing 2% (w/v) BSA (vehicle) ± 700 μM (B and C) oleic acid (OA) or (D and E) linoleic acid (LOA) for 16h prior to stimulation with insulin (20 nM, 10min). Cells were washed to remove insulin and subsequently incubated with 100 nM wortmannin (WM) and lysed at times indicated. Immunoblots and relevant quantification of phospho-Akt and phospho-ERK1/2 in L6 myotubes incubated with OA (B,C) or LOA (D,E). Data in the bar graphs is presented as mean ± SEM (n = 3) with asterisks indicating a significant change (p<0.05) between the respective filled (fatty acid treatment) and unfilled (control) bar at each time point.</p

Effects of insulin, oleic acid (OA) and linoleic acid (LOA) on Akt and ERK phosphorylation in rat L6 myotubes.

<p>(<b>A</b>) L6 myotubes were serum-starved for 2h prior to stimulation with insulin (10 min) at concentrations indicated. (<b>B,C</b>) L6 myotubes were incubated with serum-free media containing 2% (w/v) BSA (vehicle) ± (<b>B</b>) oleic acid (OA) or (C) linoleic acid (LOA) at concentrations indicated, for 16h prior to stimulation with insulin (20 nM, 10min). (D,E) L6 myotubes were incubated as in (<b>B</b>) ± 700 μM (<b>D</b>) OA or (<b>E</b>) LOA for times indicated, prior to stimulation with insulin (20 nM, 10min). (A-E) Cell lysates were immunoblotted using antibodies against the proteins indicated. The asterisks signify a significant change between the indicated bars (p<0.05).</p

Effects of insulin, palmitate (PA), oleic acid (OA) and linoleic acid (LOA) on Akt, Src and PP2Ac phosphorylation and PP2Ac demethylation in L6 myotubes.

<p>L6 myotubes were incubated with serum-free media containing 2% (w/v) BSA (vehicle) ± 700 μM palmitate (PA), oleic acid (OA) or linoleic (LOA) for 16h or for times indicated. In some experiments, cells were treated with fatty acids either alone or in combination as indicated or stimulated with insulin (20 nM, 10min) as indicated (<b>A-E</b>). Cell lysates were immunoblotted using antibodies against the proteins/phospho-proteins indicated. In (<b>E</b>) PP2Ac was immunoprecipitated from cell lysates and the immunopellet probed with an antibody recognising demethylated PP2Ac. Bar graphs values (A, n = 3; B, n = 4 and E, n = 7) are Mean ± SEM. The asterisks in (<b>A</b>) signify a significant change (p<0.05) compared to the insulin treated value (Lane 2) or between the indicated bars (<b>B & E</b>).</p

Sustained Action of Ceramide on the Insulin Signaling Pathway in Muscle Cells: IMPLICATION OF THE DOUBLE-STRANDED RNA-ACTIVATED PROTEIN KINASE*

International audienceIn vivo, ectopic accumulation of fatty acids in muscles leads to alterations in insulin signaling at both the IRS1 and Akt steps. However, in vitro treatments with saturated fatty acids or their derivative ceramide demonstrate an effect only at the Akt step. In this study, we adapted our experimental procedures to mimic the in vivo situation and show that the double-stranded RNA-dependent protein kinase (PKR) is involved in the long-term effects of saturated fatty acids on IRS1. C2C12 or human muscle cells were incubated with palmitate or directly with ceramide for short or long periods, and insulin signaling pathway activity was evaluated. PKR involvement was assessed through pharmacological and genetic studies. Short-term treatments of myotubes with palmitate, a ceramide precursor, or directly with ceramide induce an inhibition of Akt, whereas prolonged periods of treatment show an additive inhibition of insulin signaling through increased IRS1 serine 307 phosphorylation. PKR mRNA, protein, and phosphorylation are increased in insulin-resistant muscles. When PKR activity is reduced (siRNA or a pharmacological inhibitor), serine phosphorylation of IRS1 is reduced, and insulin-induced phosphorylation of Akt is improved. Finally, we show that JNK mediates ceramide-activated PKR inhibitory action on IRS1. Together, in the long term, our results show that ceramide acts at two distinct levels of the insulin signaling pathway (IRS1 and Akt). PKR, which is induced by both inflammation signals and ceramide, could play a major role in the development of insulin resistance in muscle cells