Adenovirus-mediated overexpression of human apolipoprotein E (apoE) induces hyperlipidemia by stimulating the VLDL-triglyceride (TG) production rate and inhibiting the LPL-mediated VLDL-TG hydrolysis rate. Because apoC-III is a strong inhibitor of TG hydrolysis, we questioned whether Apoc3 deficiency might prevent the hyperlipidemia induced by apoE overexpression in vivo. Injection of 2 x 10(9) plaque-forming units of AdAPOE4 caused severe combined hyperlipidemia in Apoe-/- mice [TG from 0.7 +/- 0.2 to 57.2 +/- 6.7 mM; total cholesterol (TC) from 17.4 +/- 3.7 to 29.0 +/- 4.1 mM] that was confined to VLDL/intermediate density lipoprotein-sized lipoproteins. In contrast, Apoc3 deficiency resulted in a gene dose-dependent reduction of the apoE4-associated hyperlipidemia (TG from 57.2 +/- 6.7 mM to 21.2 +/- 18.5 and 1.5 +/- 1.4 mM; TC from 29.0 +/- 4.1 to 16.4 +/- 9.8 and 2.3 +/- 1.8 mM in Apoe-/-, Apoe-/-.Apoc3+/-, and Apoe-/-.Apoc3-/- mice, respectively). In both Apoe-/- mice and Apoe-/-.Apoc3-/- mice, injection of increasing doses of AdAPOE4 resulted in up to a 10-fold increased VLDL-TG production rate. However, Apoc3 deficiency resulted in a significant increase in the uptake of TG-derived fatty acids from VLDL-like emulsion particles by white adipose tissue, indicating enhanced LPL activity. In vitro experiments showed that apoC-III is a more specific inhibitor of LPL activity than is apoE. Thus, Apoc3 deficiency can prevent apoE-induced hyperlipidemia associated with a 10-fold increased hepatic VLDL-TG production rate, most likely by alleviating the apoE-induced inhibition of VLDL-TG hydrolysis.