We have examined hepatic, genomic, and metabolic responses to dietary protein restriction in the non-pregnant Sprague-Dawley rat. Animals were pair-fed either a 6 or 24% casein-based diet for 7-10 days. At the end of the dietary period, a microarray analysis of the liver was performed, followed by validation of the genes of interest. The rates of appearance of phenylalanine, methionine, serine, and glucose and the contribution of pyruvate to serine and glucose were quantified using tracer methods. Plasma and tissue amino acid levels, enzyme activities, and metabolic intermediates were measured. Protein restriction resulted in significant differential expression of a number of genes involved in cell cycle, cell differentiation, transport, transcription, and metabolic processes. RT-PCR showed that the expression of genes involved in serine biosynthesis and fatty acid oxidation was higher, and those involved in fatty acid synthesis and urea synthesis were lower in the liver of protein-restricted animals. Free serine and glycine levels were higher and taurine levels lower in all tissues examined. Tracer isotope studies showed an ϳ50% increase in serine de novo synthesis. Pyruvate was the primary (ϳ90%) source of serine in both groups. Transmethylation of methionine was significantly higher in the protein-restricted group. This was associated with a higher S-adenosylmethionine/Sadenosylhomocysteine ratio and lower cystathione ␤-synthase and cystathionine ␥-lyase activity. Dietary isocaloric protein restriction results in profound changes in hepatic one-carbon metabolism within a short period. These may be related to high methylation demands placed on the organism and caused by possible changes in cellular osmolarity as a result of the efflux of the intracellular taurine.The metabolic and biochemical impact of qualitative and quantitative changes in dietary protein intake continues to be of interest because of their implications for public health and because of a number of studies showing a strong relationship between protein intake during pregnancy with cardiovascular function, hypertension, and glucose intolerance in the offspring (1-3). Studies in humans, primarily focused on whole body protein, nitrogen metabolism, and protein accretion, show that both high and low protein intake can impact these processes (4 -7). However, how humans adapt to protein deprivation or the biochemical mechanisms involved has not been examined. In the context of pregnancy, both high protein intake (in humans) and protein restriction in rodents have been shown to cause fetal metabolic programming and consequently altered physiological response in the offspring in adulthood (8 -11). In the rodent, dietary protein restriction during pregnancy results in growth retardation, impaired beta cell function and mass, impaired insulin sensitivity, hypertension, and other pathological responses in the offspring (1, 2). These have been associated with a change in the hypothalamic-pituitary-adrenal axis, changes in the renin-angiotensin system, and ...