Prolactin-mediated regulation of lipid biosynthesis genes in vivo in the lactating mammary epithelial cell

Abstract

Prolactin (PRL) is known to play an essential role in mammary alveolar proliferation in the pregnant mouse, but its role in lactation has been more difficult to define. Genetic manipulations that alter expression of the PRL receptor and its downstream signaling molecules resulted in developmental defects that may directly or indirectly impact secretory activation and lactation. To examine the in vivo role of PRL specifically in lactation, bromocriptine (BrCr) was administered every 8 h to lactating mice on the second day postpartum, resulting in an ~95% decrease in serum PRL levels. Although morphological changes in secretory alveoli were slight, by 8 h of BrCr, pup growth was inhibited significantly. Phosphorylated STAT5 fell to undetectable levels within 4 h. Decreased milk protein gene expression, β-casein, and α-lactalbumin, was observed after 8 h of treatment. To assess mammary-specific effects on lipid synthesis genes, we isolated mammary epithelial cells (MECs) depleted of mammary adipocytes. Expression of genes involved in glucose uptake, glycolysis, pentose phosphate shunt, de novo synthesis of fatty acids, and biosynthesis of triacylglycerides was decreased up to 19-fold in MECs by just 8 h of BrCr treatment. Glands from BrCr-treated mice showed a twofold reduction in intracellular cytoplasmic lipid droplets and a reduction in cytosolic β-casein. These data demonstrate that PRL signaling regulates MEC-specific lipogenic gene expression and that PRL signals coordinate the milk synthesis and mammary epithelial cell survival during lactation in the mouse.

Fig. 1.

Effect of bromocriptine (BrCr) treatment on levels of serum prolactin (PRL) and growth of pups. A: serum was collected from controls or mice injected with BrCr every 8 h at indicated times (filled bars). Blood samples used for quantitation of serum PRL were drawn immediately prior to subsequent BrCr injection, demonstrating that this treatment regimen successfully inhibits pituitary secretion of PRL during the entire 8 h following treatment with BrCr. For comparison, serum was also collected from dams in which the pups had been withdrawn at time 0 (gray bars). Mean serum levels of PRL ± SE are shown; n = 4 mice per time point. B: litters were standardized to 8 pups and weighed at 4, 8, 16, and 24 h. Control mice were not injected with BrCr (●), and test mice were injected with BrCr every 8 h (○). One set of mice was injected with BrCr at time 0 and then allowed to recover without additional treatment (▴). Each point represents mean increase in weight as a %time 0 weight of that litter ± SE; n = 4 at 4 h, 15 at 8 h, 8 at 16 h, and 4 at 24 h; n = 3 for BrCr recovery study. Error bars indicate SE.

Fig. 2.

BrCr-induced changes in mammary gland morphology. Representative images from hematoxylin and eosin-stained sections from mammary glands of control and BrCr-injected mice. A: control mice on day 2 of lactation. B: 4 h postinjection with BrCr. C: 8 h postinjection. D: 16 h postinjection. E: 24 h postinjection. F: untreated control mice on day 3 of lactation. *, adipose tissue; arrows, lipid droplets in lumina; arrowheads, luminal apoptotic cells. Bar = 200 μm.

Fig. 3.

Oxytocin does not restore pup weight gain in BrCr-treated mice. Mice were treated with BrCr or vehicle (control) at time 0, and pups were removed for 2 h. All mice were then injected with 300 μl 2 U/ml oxytocin, and pups returned and were allowed to suckle for another 4 h. Pups were then weighed again to determine weight gain during the 4-h suckling period. Error bars indicate SE; n = 3 for both BrCr and control groups.

Fig. 4.

BrCr treatment inhibits phosphorylation of STAT5 but not ERK1/2. A: lysates were prepared from mammary glands isolated from mice treated with BrCr 0, 4, 8, 16, and 24 h after initial treatment with BrCr. Treatment with BrCr was repeated at 8 and 16 h to maintain low serum levels of PRL. Immunoblots were probed with anti-phospho-STAT5, anti-STAT5, anti-phospho-ERK, anti-ERK, and anti-actin to demonstrate equal phospho-STAT5 and phospho-ERK. Blots show representative data from individual mice. B: graphs show quantitative data from 4 mice per experiment.

Fig. 5.

BrCr treatment decreases expression of milk protein and lipogenic genes in whole mammary gland. Standard curve method qRT-PCR was used to determine gene expression profiles for milk proteins CSN2 and WAP as well as for Glut1, mitochondrial citrate transporter (Slc25a1), de novo fatty acid synthesis enzymes Acly and Fasn, and regulatory factors Srebp1c and Akt1. Profiles are expressed as transcript copy number detected in 50 ng of total RNA relative to 0 time point and error bars are SE for 4 animals per time point. *Means differ significantly (P ≤ 0.05) from control.

Fig. 6.

Cytoplasmic secretory product are reduced following 8 h of BrCr treatment. A: histological sections of mammary glands from control (top) and 8-h BrCr-treated mice (bottom) were stained as described in materials and methods with anti-adipophilin to outline cytoplasmic lipid droplets (CLD; red), an anti-casein antibody, to show casein distribution (green), and DAPI (blue) to identify nuclei of stained cells. Light dotted box at left denotes magnified area at right. White arrows point to CLDs; arrowheads denote intracytoplasmic casein aggregates, likely in Golgi and secretory vesicles. B: CLD number was quantified and shown as the ratio to alveolar nuclei; a twofold decrease in CLD per cell was observed in mammary glands of BrCr-treated mice. C: epithelial width was decreased 35% in these sections. For B and C, data from 3 sections per mouse were averaged from Suppl. Fig. S1, in which it is possible to denote the extent of the cytoplasm from the autofluorescences of these secretory cells. Error bars show SE when data from 3 mice per condition were averaged.