To investigate the effect of a sustained (7-day) decrease in plasma free fatty acid (FFA) concentrations on insulin action and intramyocellular long-chain fatty acyl-CoAs (LCFA-CoAs), we studied the effect of acipimox, a potent inhibitor of lipolysis, in seven type 2 diabetic patients (age 53 +/- 3 years, BMI 30.2 +/- 2.0 kg/m2, fasting plasma glucose 8.5 +/- 0.8 mmol/l, HbA 1c 7.5 +/- 0.4%). Subjects received an oral glucose tolerance test (OGTT) and 120-min euglycemic insulin (80 mU/m2 per min) clamp with 3-[3H]glucose/vastus lateralis muscle biopsies to quantitate rates of insulin-mediated whole-body glucose disposal (Rd) and intramyocellular LCFA-CoAs before and after acipimox (250 mg every 6 h for 7 days). Acipimox significantly reduced fasting plasma FFAs (from 563 +/- 74 to 230 +/- 33 micromol/l; P < 0.01) and mean plasma FFAs during the OGTT (from 409 +/- 44 to 184 +/- 22 micromol/l; P < 0.01). After acipimox, decreases were seen in fasting plasma insulin (from 78 +/- 18 to 42 +/- 6 pmol/l; P < 0.05), fasting plasma glucose (from 8.5 +/- 0.8 to 7.0 +/- 0.5 mmol/l; P < 0.02), and mean plasma glucose during the OGTT (from 14.5 +/- 0.8 to 13.0 +/- 0.8 mmol/l; P < 0.05). After acipimox, insulin-stimulated Rd increased from 3.3 +/- 0.4 to 4.4 +/- 0.4 mg x kg(-1) x min(-1) (P < 0.03), whereas suppression of endogenous glucose production (EGP) was similar and virtually complete during both insulin clamp studies (0.16 +/- 0.10 vs. 0.14 +/- 0.10 mg x kg(-1) x min(-1); P > 0.05). Basal EGP did not change after acipimox (1.9 +/- 0.2 vs. 1.9 +/- 0.2 mg x kg(-1) x min(-1)). Total muscle LCFA-CoA content decreased after acipimox treatment (from 7.26 +/- 0.58 to 5.64 +/- 0.79 nmol/g; P < 0.05). Decreases were also seen in muscle palmityl CoA (16:0; from 1.06 +/- 0.10 to 0.75 +/- 0.11 nmol/g; P < 0.05), palmitoleate CoA (16:1; from 0.48 +/- 0.05 to 0.33 +/- 0.05 nmol/g; P = 0.07), oleate CoA (18:1; from 2.60 +/- 0.11 to 1.95 +/- 0.31 nmol/g; P < 0.05), linoleate CoA (18:2; from 1.81 +/- 0.26 to 1.38 +/- 0.18 nmol/g; P = 0.13), and linolenate CoA (18:3; from 0.27 +/- 0.03 to 0.19 +/- 0.02 nmol/g; P < 0.03) levels after acipimox treatment. Muscle stearate CoA (18:0) did not decrease after acipimox treatment. The increase in R(d) correlated strongly with the decrease in muscle palmityl CoA (r = 0.75, P < 0.05), oleate CoA (r = 0.76, P < 0.05), and total muscle LCFA-CoA (r = 0.74, P < 0.05) levels. Plasma adiponectin did not change significantly after acipimox treatment (7.9 +/- 1.8 vs. 7.5 +/- 1.5 microg/ml). These data demonstrate that the reduction in intramuscular LCFA-CoA content is closely associated with enhanced insulin sensitivity in muscle after a chronic reduction in plasma FFA concentrations in type 2 diabetic patients despite the lack of an effect on plasma adiponectin concentration.