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Mechanism by Which Fatty Acids Inhibit Insulin Activation of Insulin Receptor Substrate-1 (IRS-1)-associated Phosphatidylinositol 3-Kinase Activity in Muscle*

Open AccessPublished:November 14, 2002DOI:https://doi.org/10.1074/jbc.M200958200
      Recent studies have demonstrated that fatty acids induce insulin resistance in skeletal muscle by blocking insulin activation of insulin receptor substrate-1 (IRS-1)-associated phosphatidylinositol 3-kinase (PI3-kinase). To examine the mechanism by which fatty acids mediate this effect, rats were infused with either a lipid emulsion (consisting mostly of 18:2 fatty acids) or glycerol. Intracellular C18:2 CoA increased in a time-dependent fashion, reaching an ∼6-fold elevation by 5 h, whereas there was no change in the concentration of any other fatty acyl-CoAs. Diacylglycerol (DAG) also increased transiently after 3–4 h of lipid infusion. In contrast there was no increase in intracellular ceramide or triglyceride concentrations during the lipid infusion. Increases in intracellular C18:2 CoA and DAG concentration were associated with protein kinase C (PKC)-θ activation and a reduction in both insulin-stimulated IRS-1 tyrosine phosphorylation and IRS-1 associated PI3-kinase activity, which were associated with an increase in IRS-1 Ser307 phosphorylation. These data support the hypothesis that an increase in plasma fatty acid concentration results in an increase in intracellular fatty acyl-CoA and DAG concentrations, which results in activation of PKC-θ leading to increased IRS-1 Ser307 phosphorylation. This in turn leads to decreased IRS-1 tyrosine phosphorylation and decreased activation of IRS-1-associated PI3-kinase activity resulting in decreased insulin-stimulated glucose transport activity.
      IRS-1
      insulin receptor substrate-1
      IR
      insulin receptor
      PI3-kinase
      phosphatidylinositol 3-kinase
      PKC
      protein kinase C
      DAG
      diacylglycerol
      LCACoA
      long-chain acyl-CoA
      LC/MS/MS
      liquid chromatography tandem mass spectrometry
      MOPS
      4-morpholinepropanesulfonic acid
      TNFα
      tumor necrosis factor α
      Insulin resistance in skeletal muscle is a major factor in the pathogenesis of type 2 diabetes. Recent studies in animals and humans have demonstrated a strong relationship with increased intramuscular triglyceride content (
      • Kraegen E.W.
      • Cooney G.J., Ye, J.M.
      • Thompson A.L.
      • Furler S.M.
      ,
      • Kim J.K.
      • Gavrilova O.
      • Chen Y.
      • Reitman M.L.
      • Shulman G.I.
      ,
      • Kim J.K.
      • Fillmore J.J.
      • Chen Y., Yu, C.
      • Moore I.K.
      • Pypaert M.
      • Lutz E.P.
      • Kako Y.
      • Velez-Carrasco W.
      • Goldberg I.J.
      • Breslow J.L.
      • Shulman G.I.
      ,
      • Pan D.
      • Lillioja S.
      • Kriketos A.
      • Milner M.
      • Baur L.
      • Bogardus C.
      • Jenkins A.
      • Storlien L.
      ) and intramyocellular triglyceride content as assessed by 1H NMR (
      • Krssak M.
      • Falk Petersen K.
      • Dresner A.
      • DiPietro L.
      • Vogel S.M.
      • Rothman D.L.
      • Roden M.
      • Shulman G.I.
      ,
      • Perseghin G.
      • Scifo P., De
      • Cobelli F.
      • Pagliato E.
      • Battezzati A.
      • Arcelloni C.
      • Vanzulli A.
      • Testolin G.
      • Pozza G.
      • Del Maschio A.
      • Luzi L.
      ,
      • Jacob S.
      • Machann J.
      • Rett K.
      • Bretchel K.
      • Volk A.
      • Renn W.
      • Maerker E.
      • Matthaei S.
      • Schick F.
      • Claussen C.D.
      • Harring H.U.
      ). In addition, infusions of lipid emulsions with heparin to acutely raise plasma fatty acid concentrations have also been shown to cause profound insulin resistance in rat and human skeletal muscle within 4–6 h (
      • Boden G.
      • Chen X.
      ,
      • Roden M.
      • Price T.B.
      • Perseghin G.
      • Petersen K.F.
      • Rothman D.L.
      • Cline G.W.
      • Shulman G.I.
      ,
      • Dresner A.
      • Laurent D.
      • Marcucci M.
      • Griffin M.E.
      • Dufour S.
      • Cline G.
      • Slezak L.A.
      • Andersen D.K.
      • Hundal R.S.
      • Rothman D.L.
      • Petersen K.F.
      • Shulman G.I.
      ,
      • Griffin M.E.
      • Marcucci M.J.
      • Cline G.W.
      • Bell K.
      • Barucci N.
      • Lee D.
      • Goodyear L.J.
      • Kraegen E.W.
      • White M.F.
      • Shulman G.I.
      ). The mechanism by which fatty acids induce insulin resistance in skeletal muscle remains controversial. Randle et al. (
      • Randle P.J.
      • Garland P.B.
      • Hales C.N.
      • Newsholme E.A.
      ,
      • Randle P.J.
      • Newsholme E.A.
      • Garland P.B.
      ) first suggested that fatty acids might induce insulin resistance in skeletal muscle by inhibiting pyruvate dehydrogenase activity, resulting in an increase in intracellular citrate concentration, which would then result in inhibition of phosphofructokinase activity leading to an increase in intracellular glucose-6-phosphate; this in turn would inhibit hexokinase activity, resulting in decreased glucose uptake. More recent 31P/13C NMR studies in humans have revealed a very different mechanism of fatty acid-induced insulin resistance whereby an increase in plasma fatty acid concentration was shown to result in lower intramyocellullar glucose 6-phosphate (
      • Roden M.
      • Price T.B.
      • Perseghin G.
      • Petersen K.F.
      • Rothman D.L.
      • Cline G.W.
      • Shulman G.I.
      ,
      • Roden M.
      • Krssak M.
      • Stingl H.
      • Gruber S.
      • Hofer A.
      • Furnsinn C.
      • Moser E.
      • Waldhausl W.
      ) and glucose concentrations (
      • Dresner A.
      • Laurent D.
      • Marcucci M.
      • Griffin M.E.
      • Dufour S.
      • Cline G.
      • Slezak L.A.
      • Andersen D.K.
      • Hundal R.S.
      • Rothman D.L.
      • Petersen K.F.
      • Shulman G.I.
      ), suggesting that fatty acids inhibit insulin-stimulated glucose transport activity (
      • Dresner A.
      • Laurent D.
      • Marcucci M.
      • Griffin M.E.
      • Dufour S.
      • Cline G.
      • Slezak L.A.
      • Andersen D.K.
      • Hundal R.S.
      • Rothman D.L.
      • Petersen K.F.
      • Shulman G.I.
      ). These changes were associated with reduced insulin-stimulated IRS-11 tyrosine phosphorylation (
      • Griffin M.E.
      • Marcucci M.J.
      • Cline G.W.
      • Bell K.
      • Barucci N.
      • Lee D.
      • Goodyear L.J.
      • Kraegen E.W.
      • White M.F.
      • Shulman G.I.
      ) and IRS-1-associated phosphatidylinositol 3-kinase (PI3-kinase) activity (
      • Dresner A.
      • Laurent D.
      • Marcucci M.
      • Griffin M.E.
      • Dufour S.
      • Cline G.
      • Slezak L.A.
      • Andersen D.K.
      • Hundal R.S.
      • Rothman D.L.
      • Petersen K.F.
      • Shulman G.I.
      ,
      • Griffin M.E.
      • Marcucci M.J.
      • Cline G.W.
      • Bell K.
      • Barucci N.
      • Lee D.
      • Goodyear L.J.
      • Kraegen E.W.
      • White M.F.
      • Shulman G.I.
      ) suggesting that fatty acids cause insulin resistance through inhibition of insulin signaling, which we hypothesized might occur through activation of a serine kinase cascade involving PKC-θ (
      • Griffin M.E.
      • Marcucci M.J.
      • Cline G.W.
      • Bell K.
      • Barucci N.
      • Lee D.
      • Goodyear L.J.
      • Kraegen E.W.
      • White M.F.
      • Shulman G.I.
      ). To explore the possible roles of different intracellular fatty acid metabolites such as fatty acyl-CoA, diacylglycerol (DAG), ceramides, and triglycerides in mediating fatty acid-induced insulin resistance in skeletal muscle, we measured these metabolites at different time intervals during a lipid infusion in relation to insulin stimulation: (i) insulin receptor tyrosine phosphorylation, (ii) IRS-1 tyrosine phosphorylation, and (iii) IRS-1-associated PI3-kinase activity as well as PKC-θ translocation. In a separate group of in vitro soleus muscle studies, we also examined whether fatty acid-induced defects in insulin signaling were coupled to defects in insulin-stimulated glucose uptake across a range of insulin concentrations.

      RESULTS

      Basal plasma fatty concentration increased rapidly following the lipid/heparin infusion and remained constant until the saline wash-out period during which time it returned to base-line concentration (Fig. 1 A). This increase in plasma fatty acid concentration in the lipid-infused group resulted in increases in both intramuscular LCACoAs and DAG concentration in the soleus muscle compared with the control group (Fig. 1, B andC). Although the LCACoA continued to increase throughout the lipid infusion, the DAGs reached a peak concentration at 3–4 h and then surprisingly decreased to basal concentrations despite continued lipid infusion (Fig. 1 C). In contrast, lipid infusion had no effect on intramyocellullar ceramide content (Fig. 1 D) or muscle triglyceride (Fig. 1 E) content except at the 1-h time point, at which time the concentration decreased compared with base line. The increase in total LCACoA concentration could be accounted for entirely by a selective increase in C18:2 CoA (major fatty acid composition in liposyn II) (3.86 ± 0.46 nmol/g of weight for control group, 9.30* ± 0.87, 16.17** ± 2.37, and 18.89** ± 2.51 nmol/g of weight after a 1-h, 3-h, and 5-h lipid infusion and 7.22 ± 1.22 nmol/g of weight after wash-out period; *, p < 0.05 versus control; **,p < 0.001 versus control; Fig. 2 A). In contrast the transient ∼3–4-fold increase in total DAG content at 3–4 h (0.65 ± 0.14 μmol/g of weight for control group, 1.43 ± 0.51, 2.73 ± 0.83+, 2.54 ± 0.79+, 1.36 ± 0.40, 0.96 ± 0.31 μmol/g of weight for 1-h, 3-h, 4-h, 5-h and wash-out groups, respectively; +, p ≤ 0.006versus control) could be attributed to an increase in virtually all DAG species (Fig. 2 B). These increases in intracellular LCACoA and DAG concentrations were associated with PKC-θ activation, as reflected by a significant reduction in the fraction of PKC-θ in the cytosol and a significant increase in the PKC-θ membrane-associated/cytosol fraction after 5 h of lipid infusion (both p = 0.04 versus control group; Fig. 3). There was also a reduction in total PKC-θ content, which is consistent with previous observations in a high-fat fed rat model that had increased intramuscular lipid accumulation (
      • Schmitz-Peiffer C.
      • Browne C.L.
      • Oakes N.D.
      • Watkinson A.
      • Chisholm D.J.
      • Kraegen E.W.
      • Biden T.J.
      ).
      Figure thumbnail gr1
      Figure 1Time course for plasma fatty acid and intracellular fat metabolite concentrations in soleus muscles during lipid infusion. A, plasma fatty acid concentrations;B, LCACoA concentrations; C, diacylglyceride concentrations; D, ceramide concentrations; E, triglyceride concentrations. Values are means ± S.E. for 6–10 experiments. *, p < 0.05 versuscontrol groups; +, p ≤ 0.006, and **,p < 0.001 versus base line.
      Figure thumbnail gr2
      Figure 2Time course for the concentration profiles of LCACoAs and DAG in soleus muscles during the lipid infusion. A, individual LCACoAs species were quantitated:C16:1, palmitoleoyl-CoA; C16:0, palmitoyl-CoA;C18:2, linoleoyl-CoA; C18:1, oleoyl-CoA; andC18:0, stearoyl-CoA. Values are means ± S.E. for 6–10 experiments. *, p < 0.05 versus control group; **, p < 0.001 versus control group.B, DAG species were abbreviated as two contributing fatty acyl groups. S, stearoyl; O, oleoyl;L, linoleoyl; P, palmitoyl; Po, palmitoleoyl. Values are means ± S.E. for 3–9 experiments. *,p < 0.05 versus control group.
      Figure thumbnail gr3
      Figure 3Time course for the effects of fatty acids on PKC -θ activity in soleus muscle in vivo. PKC-θ protein levels were determined in the cytosolic and membrane fraction by immunoblotting with PKC-θ specific antibodies. Total PKC-θ levels were calculated from the sum of cytosolic and membrane-associated amounts, and PKC-θ distribution was expressed as the ratio of membrane-associated to cytosolic amounts.W/O, without. Values are means ± S.E. for 6–10 experiments. *, p < 0.05 versus control groups.
      The increase in intracellular fatty acyl-CoA and PKC-θ activation were also associated with a significant impairment in insulin-stimulated IRS-1 tyrosine phosphorylation and IRS-1-associated PI3-kinase activity after 5 h of lipid infusion (Fig. 4). These changes were associated with a 1.6-fold increase (p = 0.002 versus control) in IRS-1 Ser307 phosphorylation following 5 h of lipid infusion (Fig. 5). In contrast lipid infusion did not inhibit insulin-stimulated insulin receptor tyrosine phosphorylation (Fig. 4).
      Figure thumbnail gr4
      Figure 4Time course for the effects of fatty acids on insulin signaling in soleus muscle in vivo.Insulin-stimulated IR tyrosine phosphorylation, insulin-stimulated IRS-1 tyrosine phosphorylation, and insulin-stimulated IRS-1-associated PI3-kinase activity are all expressed as fold increase of insulin stimulation over basal states. Values are means ± S.E. for 5–8 experiments. *, p < 0.05 versus control groups.
      Figure thumbnail gr5
      Figure 5Effects of fatty acids on IRS-1 Ser307 phosphorylation in soleus muscle in vivo. A, IRS-1 Ser307phosphorylation was detected with a polyclonal antibody raised specifically for phosphorylated Ser307 (upper panel). Nitrocellulose membranes were stripped and reprobed with IRS-1 antibody to ensure equal amount of protein loading (lower panel). IP, immunoprecipitate; INS, insulin; Gly, glycerol; WB, Western blot.B, degree of IRS-1 Ser307 phosphorylation in glycerol- and lipid-infused groups. Values are means ± S.E. from six rats for each group. *, p < 0.05 versusglycerol-infused rats.
      Following the 3-h lipid wash-out period, intracellular 18:2 acyl-CoA returned to base-line concentrations, and PKC-θ activity returned to normal (Figs. 1 and 3). In parallel with these results insulin-stimulated IRS-1 tyrosine phosphorylation and IRS-1-associated PI3-kinase activity also returned to normal.
      To determine whether higher concentrations of insulin could overcome these lipid-induced defects in insulin signaling and action, we also examined insulin-stimulated muscle glucose uptake and insulin signaling across a wide range of insulin concentrations (50, 1,000, and 10,000 microunits/ml) in an in vitro soleus muscle preparation following 5 h of either lipid or glycerol infusion. Consistent with our previous results, 5 h of lipid infusion induced a profound defect in insulin-stimulated glucose uptake, which occurred across all insulin concentrations (Fig. 6). This reduction in insulin-stimulated glucose uptake was paralleled by similar reductions in insulin-stimulated IRS-1 tyrosine phosphorylation and IRS-1-associated PI3-kinase activity across all insulin concentrations, but there was no change in insulin receptor tyrosine phosphorylation (Fig. 7). Taken together these results demonstrates that fatty acids induce a defect in insulin activation of PI3-kinase at the level of IRS-1 tyrosine phosphorylation that cannot be overcome with supraphysiologic concentrations of insulin.
      Figure thumbnail gr6
      Figure 6Insulin dose response for the effects of fatty acids on insulin-stimulated 2-deoxyglucose uptake in soleus muscle in vitro. Soleus muscles were isolated from glycerol- or lipid-infused rats. They were then incubated with insulin at 0, 50, 1000, and 10,000 microunits/ml. The rate of 2-deoxyglucose uptake was measured and expressed as fold change over non-insulin-stimulated groups. Values are means ± S.E. from 6–9 experiments. *, p < 0.05 versusglycerol-infused rats.
      Figure thumbnail gr7
      Figure 7Insulin dose response for the effects of fatty acids on insulin signaling in soleus muscle in vitro. A, IR tyrosine phosphorylation was detected with phosphotyrosine-specific antibody (upper panel). Nitrocellulose membranes were stripped and reprobed with insulin receptor antibody to ensure equal amount of protein loading (lower panel). The bar graph shows the degree of IR tyrosine phosphorylation in glycerol- or lipid-infused soleus musclein vitro. IP, immunoprecipitate.B, IRS-1 tyrosine phosphorylation was detected with phosphotyrosine-specific antibody (P-tyr, upper panel). Nitrocellulose membranes were stripped and reprobed with IRS-1 antibody to ensure equal amounts of protein loading (lower panel). The bar graph shows the degree of IRS-1 tyrosine phosphorylation in glycerol- or liposyn-infused soleus musclein vitro. WB, Western blot. C, IRS-1-associated PI3-kinase activity was detected by measuring32P incorporation into phosphatidylinositol (PI3P). The bar graph shows the IRS-1-associated PI3-kinase activity in glycerol- or liposyn-infused soleus muscle in vitro. Values are means ± S.E. from eight independent experiments. *, p < 0.05versus glycerol-infused rats.

      DISCUSSION

      To examine the possible roles of fatty acyl-CoA, diacylglycerol, ceramides, and triglycerides in mediating fatty acid induced insulin resistance in skeletal muscle, we assessed the intracellular concentration of these metabolites at different time intervals during a lipid infusion in awake rats. The changes in these fatty acid metabolite concentrations were then compared with changes in insulin-stimulated insulin receptor tyrosine phosphorylation, IRS-1 tyrosine phosphorylation, IRS-1-associated PI3-kinase activity, and PKC-θ translocation. We found that during the lipid infusion intra- myocellullar C18:2 CoA concentration increased by ∼6-fold and that it was the only intracellular fatty acyl-CoA to increase. Because the infused intralipid consisted mostly of C18:2 fatty acids, these data strongly suggest that this intracellular fatty acyl-CoA was derived from the infused lipid. Following the increase in intracellular C18:2 CoA, there was a ∼3-fold increase in intracellular DAG, which peaked at 3–4 h and then surprisingly declined despite persistent elevation in plasma fatty acid concentrations. In contrast to the fatty acyl-CoA, which consisted mostly of C18:2 fatty acids, the increase in DAG consisted of virtually all measured fatty acids. Taken together these data suggest that an increase in intracellular fatty acyl-CoA activates a phospholipase that leads to production of DAG from endogenous lipid sources, which might explain the observed decrease in intramuscular triglyceride content during the first couple of hours of the lipid infusion. In contrast to the increases in intracellular fatty acyl-CoA and DAG, there were no significant increases in intracellular ceramides or triglyceride concentrations during the 5-h lipid infusion, which suggests that these metabolites do not play a major role in mediating fatty acid-induced insulin resistance in skeletal muscle.
      In parallel with the increases in intracellular fatty acyl-CoA, we observed a ∼30% reduction in insulin activation of IRS-1 tyrosine phosphorylation and an ∼50% reduction in IRS-1-associated PI3-kinase activity after 5 h of lipid infusion, which coincided with activation of PKC-θ. These data might explain the 3–5 h delay for fatty acid-induced insulin resistance in skeletal muscle resulting from an intralipid/heparin infusion (
      • Boden G.
      • Chen X.
      ,
      • Roden M.
      • Price T.B.
      • Perseghin G.
      • Petersen K.F.
      • Rothman D.L.
      • Cline G.W.
      • Shulman G.I.
      ). In contrast, the lipid infusion had no effect on insulin receptor tyrosine phosphorylation. Overall these data demonstrate that increases in plasma fatty acid concentration inhibit insulin activation of IRS-1-associated PI3-kinase at the level of IRS-1, possibly though activation of PKC-θ, a known serine kinase. To gain further insights into this mechanism we assessed IRS-1 Ser307 phosphorylation. Previous in vitrostudies by Aguirre et al. (
      • Aguirre V.
      • Uchida T.
      • Yenush L.
      • Davis R.
      • White M.
      ) demonstrated that IRS-1 Ser307 phosphorylation is a critical site in mediating TNFα-induced insulin resistance in Chinese hamster ovary cells. When IRS-1 Ser307 was mutated to IRS-1 Ala307, these cells were protected from TNFα-induced insulin resistance. Indeed, in the present study we found that after 5 h of lipid infusion there was a 1.6-fold increase in IRS-1 Ser307 phosphorylation in soleus muscle, which suggests that fatty acids may mediate insulin resistance through the same common final pathway as TNFα (
      • Hotamisligil G.S.
      • Peraldi P.
      • Budavari A.
      • Ellis R.
      • White M.F.
      • Spiegelman B.M.
      ).
      To determine whether higher concentrations of insulin could overcome these fatty acid-induced defects in insulin signaling and action, we also examined these parameters in vitro, across a wide range of insulin concentrations, in soleus muscles obtained from rats following 5 h of either lipid or glycerol infusion. Consistent with our current and previous in vivo results, 5 h of lipid infusion induced a profound defect in insulin-stimulated glucose uptake (
      • Roden M.
      • Price T.B.
      • Perseghin G.
      • Petersen K.F.
      • Rothman D.L.
      • Cline G.W.
      • Shulman G.I.
      ,
      • Dresner A.
      • Laurent D.
      • Marcucci M.
      • Griffin M.E.
      • Dufour S.
      • Cline G.
      • Slezak L.A.
      • Andersen D.K.
      • Hundal R.S.
      • Rothman D.L.
      • Petersen K.F.
      • Shulman G.I.
      ,
      • Griffin M.E.
      • Marcucci M.J.
      • Cline G.W.
      • Bell K.
      • Barucci N.
      • Lee D.
      • Goodyear L.J.
      • Kraegen E.W.
      • White M.F.
      • Shulman G.I.
      ), which occurred across all insulin concentrations. This reduction in insulin-stimulated glucose uptake was paralleled by similar reductions in insulin-stimulated IRS-1 tyrosine phosphorylation and IRS-1-associated PI3-kinase activity across all insulin concentrations, but there was no change in insulin-stimulated IR tyrosine phosphorylation. Taken together these results demonstrate that the fatty acid-induced inhibition of insulin-stimulated glucose transport activity in muscle can be explained for the most part by decreased activation of PI3-kinase at the level of IRS-1 tyrosine phosphorylation, which cannot be overcome with supraphysiologic concentrations of insulin.
      In conclusion, these data provide new insights into the pathogenesis of fat-induced insulin resistance in skeletal muscle and support the hypothesis that an increase in plasma fatty acid concentration results in an increase in intracellular fatty acyl-CoA and DAG concentrations, which then results in activation of PKC-θ leading to increased IRS-1 Ser307 phosphorylation. These changes in turn result in decreased IRS-1 tyrosine phosphorylation and decreased activation of IRS-1-associated PI3-kinase, resulting in decreased insulin-stimulated glucose transport activity.

      Acknowledgments

      We acknowledge the expert technical assistance of Hyegeong Kim, Lynn Croft, Anthony Romanelli, Taca Higashimori, and Theresa Choi.

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