Understanding the effects of preconditioning on cellular metabolism is important in providing insight into how cells and tissues may behave when confronted with alterations in their environment. Previously, elevated accumulation of lipids and a correspondingly reduced rate of urea synthesis were found in primary heptocytes preconditioned in culture medium containing high levels of insulin and then exposed to plasma. Subsequent studies found that preconditioning primary hepatocytes in medium containing low levels of insulin before exposure to plasma supplemented with amino acids was able to confer resistance to the lipid-accumulating effect of plasma exposure and restored urea synthesis. In the current study, we investigated the effects of insulin preconditioning and amino acid supplementation on the gene expression profile of the urea cycle and fatty acid metabolism enzymes. We found that insulin preconditioning mediates the effects of amino acids in the plasma exposure period on urea synthesis. Urea synthesis is regulated by insulin and amino acids through both short-term and long-term control. Possible mechanisms of long-term control through transcriptional regulation of urea synthesis by insulin, amino acids, and enzymes and transcription factors associated with lipid metabolism are discussed.