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Triglycerides (TG) are synthesized in the liver principally from two sources of fatty acids (FA): FA synthesized de novo in the liver and preformed FA. We have measured the rate of secretion of de novo synthesized FA and total secretion of FA bound to VLDL-TG in healthy men (n = 5) in the basal state, and after 1 (day 1) and 4 d (day 4) of a hypercaloric carbohydrate diet (approximately 2.5 times energy expenditure) that generated a moderate endogenous hyperinsulinemia (plasma insulin approximately 60 microU/ml). Prolonged carbohydrate hyperalimentation/hyperinsulinemia increased plasma VLDL-TG approximately 10-fold in part due to a 3.4-fold increase in total VLDL-TG secretion rate (basal state = 72+/-23, day 4 = 242+/-78 micromol TG/kg/d). Although the secretion of de novo synthesized FA increased throughout the study (basal state = 1.1+/-0.4, day 1 = 15.9+/-7.9, day 4 = 50.0+/-18.8 micromol TG/ kg/d), the 2.7-fold increase in secretion rate of preformed FA (basal state = 70+/-23, day 4 = 191+/-57 micromol TG/kg/d) quantitatively contributed the most to total VLDL-TG secretion rate. Decreased catabolism of VLDL-TG also contributed to the hypertriglyceridemia as reflected by an approximately fourfold decrease in both fractional turnover rate (basal state = 9.2+/-3.8, day 1 = 2.1+/-0.2, day 4 = 2.1+/-0.3 pools/d) and rate of clearance (basal state = 0.35+/-0.08, day 1 = 0.11+/-0.01, day 4 = 0.09+/-0.01 liter/kg/d) of VLDL-TG. Thus, the primary difference between 1 and 4 d of hyperinsulinemia in conjunction with carbohydrate hyperalimentation is the increase in hepatic secretion of preformed FA into VLDL-TG.