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
). 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
< 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
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.
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
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
). 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.