Background:  Some protease inhibitors (PI) are known to increase secretion of triglycerides (apolipoprotein B) by hepatocytes, but only in the presence of oleic acid, which stimulates neutral-lipid biosynthesis, suggesting multiple cellular targets in hepatocytes. To better characterize PI effects on the cellular metabolic state, we report the hepatic metabolome, a comprehensive quantification of intracellular metabolites.

Methods:  We exposed human hepatoma (HepG2) cells for 16 hours to lopinavir (LPV), indinavir (IDV), ritonavir (RTV), and atazanavir (ATV) at 30 mM and nelfinavir (NFV) at 10 mM. All cellular metabolites were analyzed by gas or liquid chromatography mass spectrometry. The expression of each endogenous metabolite was analyzed using pattern-recognition tools using a statistical process control method (Spotfire). Treatment comparisons were by ANOVA.

Results:  Of a total of 220 metbolites measured, the intracellular concentrations of 72 (33%) were significantly increased and 26 (12%) were down-regulated by 1 or more of the PI tested (p <0.05). The up-regulated metabolites were:  amino acids; fatty acids, including oleic acid (increased 127.1± 8.5% by IDV, LPV, NFV, and RTV); and intermediates of the citrate cycle (ATP generation) and gluconeogenesis. Down-regulated metabolites included adenine (precursor of ATP) and creatinine (precursors of urea cycle). Placed in order, from the greatest to the least number of metabolites significantly up-regulated, they are:  LPV (56)> IDV (28) > NFV (24) > RTV (15) >> ATV (0).

Conclusions:  An increase in intracellular oleic acid, sugars, and amino acids by some PI is indicative of a biochemical signature of stimulated metabolism necessary for secretion and export of lipids including ApoB. Our data provide additional qualitative and quantitative data for differential regulation of lipid synthesis by individual PI.