The housekeeping genes GAPDH and cyclophilin are regulated by metabolic state in the liver of dairy cows

Steady-state levels of mRNA are often used to infer treatment effects on the levels of the corresponding protein. In addition, an internal standard RNA is usually measured to document specificity of treatment and to correct for intersample variation. Our objective was to evaluate whether glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and cyclophilin could be used as internal standards when studying changes in hepatic gene expression in dairy cows. Hepatic expression of GAPDH and cyclophilin was measured in 6 cows in late pregnancy (28 d prepartum) and early lactation (10 d postpartum). Each gene displayed 2- to 3-fold higher expression in early lactation than in late pregnancy. Next, we determined whether negative energy balance alone or in combination with exogenous growth hormone could mimic the effects of early lactation. Late-lactating cows were fed 120% of predicted energy requirements or 33% of maintenance requirements. During each feeding period, cows were administered excipient or bovine somatotropin in a single-reversal design with 4-d periods separated by a 2-d interval. Underfeeding increased hepatic expression of GAPDH and cyclophilin by 1- to 2-fold, whereas bovine somatotropin had no effect. Finally, the effects of insulin were studied by performing hyperinsulinemic-euglycemic clamps in late pregnancy (28 d prepartum) and early lactation (28 d postpartum). Hyperinsulinemia reduced GAPDH expression in both states, and cyclophilin expression in early lactation. In conclusion, GAPDH and cyclophilin are regulated in the liver of dairy cows and should not be used to standardize hepatic gene expression in studies involving the transition period, undernutrition, and sustained changes in plasma insulin.