Submitted to: Experimental Biology and Medicine
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/10/2006
Publication Date: 5/1/2006
Citation: Liu, W., Capuco, A.V., Romagnolo, D. 2006. Regulation of expression of cytosolic NADP+-dependent isocitrate dehydrogenase in bovine mannary epithelium. Experimental Biology and Medicine. 231(5):599-610. Interpretive Summary: The enzyme, isocitrate dehydrogenase, catalyzes the conversion of isocitrate to a-ketoglutarate. A particular form of this enzyme (ICD2) is present in the cytoplasm, where fatty acid synthesis takes place and where this enzyme generates a cofactor (NADPH) necessary for the synthesis of fatty acids. Regulation of this enzyme in mammary secretory cells has been studied. We showed that the amount of messenger RNA (mRNA) coding for this enzyme increased two-fold during the transition from pregnancy to lactation. Using in vitro culture, we demonstrated that generation of mRNA for the enzyme is enhanced by prolactin, a hormone that plays an important role in the final transition of mammary epithelial cells to fully active milk secreting cells, and decreased by insulin, an important metabolic regulator. The MAP kinase signaling pathway is involved in many of prolactin's regulatory actions and is likewise involved in the stimulating synthesis of ICD2 mRNA. Data indicate some of the hormones involved in regulating the onset of milk production also regulator synthesis of ICD2. Other metabolic factors and extracellular signaling pathways also are involved in this regulation.
Technical Abstract: Isocitrate dehydrogenase catalyzes the conversion of isocitrate to a-ketoglutarate. This reaction requires the presence of the cofactor NADP in the cytosol. The enzymatic activity of the cytosolic NADP+-dependent isocitrate dehydrogenase (ICD2) increases dramatically in early lactation in bovine mammary gland and generates NADPH, which can be used for de novo fatty acid synthesis. In this study, we examined the changes in expression of ICD2 in late pregnancy and at various stages of lactation in bovine mammary tissue. We found that ICD2 mRNA levels were lower in late pregnancy (-20 d), but increased (2.0-fold) after parturition (14 d) and remained constant thereafter (90, 120, and 240 d). Next, we examined the regulation of ICD2 expression in cultured BME-UV bovine mammary epithelial cells. We found that fetal calf serum and insulin (INS) repressed the expression of ICD2 mRNA in a dose-dependent fashion, whereas prolactin (PRL) had a stimulatory effect on ICD2 mRNA levels. The treatment with hydrocortisone (HC) did not influence the cellular content of ICD2 transcripts. The inhibitory effects of INS on ICD2 mRNA levels were antagonized by cotreatment with PRL suggesting these peptide hormones may exert opposing effects on expression of ICD2. Cotreatment of BME-UV cells with the mitogen-activated protein kinase (MAPK) inhibitor PD98059 abrogated the stimulatory effects of PRL on ICD2 transcripts and protein suggesting the involvement of the MAPK pathway in the positive regulation of ICD2 expression. Finally, we report that a-ketoglutarate (a-KG) and palmitic acid (PA) reduced the levels of ICD2 transcripts, whereas differentiation of BME-UV cells on extracellular matrix (Matrigel) induced expression of ICD-2 and b-casein mRNA. We propose that the expression of ICD2 in bovine mammary epithelial cells is under the control of factors important in mammary gland differentiation including lactogenic hormones and extracellular matrix, as well as metabolic effectors.