To study the effect of nonesterified fatty acids (NEFAs) on uncoupling protein-2 (UCP-2) and uncoupling protein-3 (UCP-3) gene expression, a triglyceride emulsion was infused for 5 h in 14 healthy volunteers. A euglycemic-hyperinsulinemic clamp was administered concomitantly in 7 of the 14 subjects. The mRNA levels of UCP-2 and of the short (UCP-3S) and long (UCP-3L) isoforms of UCP-3 were quantified by reverse transcription-competitive polymerase chain reaction in tissue biopsies taken before and at the end of the infusion periods. Plasma NEFA concentrations increased from 362+/-52 to 989+/-157 micromol/l (P = 0.018) during triglyceride infusion. UCP-3L (8+/-1 vs. 19+/-2 amol/microg total RNA, P = 0.018) and UCP-3S (11+/-2 vs. 17+/-3 amol/microg total RNA, P = 0.027) mRNA levels increased in skeletal muscle during triglyceride infusion. UCP-3L mRNA levels were positively correlated with plasma NEFA concentrations (r = 0.53, P = 0.005) and with lipid oxidation rates (r = 0.56, P = 0.004) determined by indirect calorimetry. In contrast, the expression of UCP-2 was not affected by lipid infusion in skeletal muscle or in subcutaneous adipose tissue. During the hyperinsulinemic clamp (plasma insulin concentrations 202+/-12 pmol/l), NEFA levels (405+/-49 vs. 648+/-77 micromol/l, P = 0.063) and lipid oxidation rates (0.67+/-0.09 vs. 0.84+/-0.10 mg x kg(-1) x min(-1), P = 0.091) were not significantly increased during triglyceride infusion. Under such conditions, the induction of UCP-3L and UCP-3S mRNA expression was totally prevented (8+/-2 vs. 8+/-1 and 8 +/-2 vs. 9+/-2 amol/microg total RNA, respectively). We conclude that increased plasma NEFA levels by lipid infusion for 5 h induces the expression of UCP-3 but not UCP-2 in humans. During triglyceride infusion, physiological hyperinsulinemia appears to prevent the induction of UCP-3 mRNA levels.