The objective of this work was to integrate experimental data on the effect of insulin, EAA and acetate into a model of transcription control in the mammary cell. Current representations of milk protein synthesis consider energy and dietary protein effects separately with no regulation other than by limiting production when supply of either is short. In reality, protein synthesis is simultaneously affected by EAA, energy substrates and hormones, by upregulating the initiation and elongation phases of mRNA translation. A central element to this process is the mammalian target of rapamycin (mTOR) which transfers nutritional and hormonal signals onto translation initiation and elongation proteins that, through phosphorylation, modulate protein synthesis. Phosphorylation data for protein kinase B (Akt), AMP activated protein kinase (AMPK), mTOR, eukaryotic elongation factor binding protein (4EBP1) and eukaryotic elongation factor 2 (eEF2) from a series of experiments in MACT cells and lactogenic mammary tissue slices conducted over the past 5 years was used to build a dynamic model representing each one of the above signaling proteins in their phosphorylated and dephosphorylated states. Akt and mTOR phosphorylation reactions were defined as Michaelis-Menten, whereas AMPK, 4EBP1 and eEF2 representations used mass action kinetics. Inference was based on resampling with replacement and resulting nonparametric confidence intervals (CI). The affinity constants for insulin effects on Akt (CI = 6.6, 32.8 ug) and isoleucine on mTOR (CI = 90, 443 uM) were within physiological concentration ranges, but the rate constant for acetate effects on AMPK was null (CI = 2.71 × 10⁻⁹, 9.8 × 10⁻⁸). Sensitivity analysis coefficients indicate isoleucine was the strongest driver (0.71) of mTOR phosphorylation compared with insulin (0.01) and acetate (0.01). Phosphorylated Akt, AMPK, mTOR, 4EBP1 and eEF2 were predicted without bias and had errors of 16, 33, 29, 28 and 33%, respectively. Insulin and isoleucine effects seem to be well represented but more work is needed to improve our understanding of the role of acetate and other AA on mTOR regulation.