Mammary Fat Can Adjust Prolactin Effect on Mammary Epithelial Cells via Leptin and Estrogen

Abstract

Leptin, like estrogen, is one of the endo/paracrine factors, which are synthesized in and secreted from mature adipocytes. The roles of the mammary fat pad and mammary adipocytes in the initiation of lactation are not clear. In this study, we showed that combination of prolactin, leptin and estrogen elevated the expression of the milk protein beta-lactoglobulin. We also showed that after prolactin stimulate the secretion of leptin from the mammary fat, leptin upregulated the expression of estrogen receptor alpha in the mammary epithelial cells. Also, prolactin affected aromatase mRNA expression in the bovine mammary fat and we demonstrated that leptin and prolactin can affect cholesterol secretion from explants in culture to the medium. Therefore, we suggest that prolactin initiates estrogen expression (as represented by aromatase mRNA) in the mammary fat pad, whereas leptin stimulates estrogen receptor alpha expression in the mammary epithelial cells. We hypothesize that leptin and estrogen, secreted from the mammary fat regulate lactation after stimulation of prolactin.

Figure 1

Effects of leptin, prolactin, and estrogen on beta-lactoglobulin expression in explants from mammary glands of lactating cows. Mammary-gland explants from lactating cows were incubated for 4 days in media containing various treatments: prolactin at 1 μg mL⁻¹, leptin at 50 ng mL⁻¹, a combination of leptin and prolactin, and a combination of leptin and estrogen at 200 ng mL⁻¹. Expression of the beta-lactoglobulin gene was evaluated by real-time PCR. Leptin alone elevated beta-lactoglobulin expression to a level of 1.2 arbitrary units, a level, which was not significantly different from that with insulin alone. Medium containing prolactin elevated beta-lactoglobulin expression to a level of 1.8 arbitrary units. When leptin and prolactin together were added to the medium, the level of beta-lactoglobulin reached 2.3 arbitrary units. Addition of estrogen to medium containing leptin and prolactin augmented the beta-lactoglobulin expression to 2.9 arbitrary units. Least squares means ± SE (n = 3). Bars with different letters differ at P < .05.

Figure 2

Expression of aromatase mRNA in mammary fat explants and mammary primary culture. Expression of aromatase mRNA in fat explants (closed bars) and primary mammary cells (open bars) from a lactating cow, with various concentrations of prolactin (0.01, 0.1 and 1 mg mL⁻¹) in the medium. The expression of aromatase in cultures of primary cells did not change significantly. Significant expression of aromatase was observed in fat explants at prolactin concentrations of 1 mg mL⁻¹. Least squares means ± SE (n = 3). Bars with different letters differ at P < .05. ∗, ∗∗ means differing at P < .05 and P < .01, respectively, between treatments with mammary primary cells and fat explants.

Figure 3

Real time analysis of ER alpha in mammary explants from lactating cow after incubation with different levels of leptin. Explants from a lactating bovine mammary gland were incubated with several concentrations of leptin (10, 100, and 1000 ng mL⁻¹). ER alpha mRNA expression was up-regulated in a dose-dependent manner. At a leptin concentration of 10 ng mL⁻¹ the expression of ER alpha reached a level of 1.3 arbitrary units, at 100 ng mL⁻¹ leptin elevated ER alpha expression to 1.9 arbitrary units, and at a leptin concentration of 100 ng mL⁻¹ the ER alpha expression level reached 3 arbitrary units. Least squares means ± SE (n = 3). Bars with different letters differ at P < .05.

Figure 4

The effect of fat explants on ER alpha expression in bovine mammary gland primary cell culture. The expression of ER alpha was examined in primary culture that was incubated with mammary fat explants (closed bars) or without mammary fat explants (open bars). Four treatments were introduced: three different levels of prolactin (0.01, 0.1, and 1 μg/mL) and one combined treatment of leptin antagonist (3200 ng/mL) and 1 μg/mL prolactin. The expression of ER alpha in primary culture incubated with no fat explants did not change in the same magnitude when compared to explants incubated with fat explants. A significant difference in the expression of ER alpha between the treatments containing fat explants and those without was observed at prolactin concentrations of 0.1 and 1 μg/mL prolactin. Leptin antagonist minimized the differences between the two treatments (with fat and without). Results are least square means ± S.E.M. of four independent experiments (n = 3). ∗, ∗∗ means differing at P < .05 and P < .001, respectively, between treatments with and without fat-explants.

Figure 5

Cholesterol secretion to the medium by mammary gland explants. (a) Effect of estrogen on cholesterol secretion to the medium from mammary explants culture. Bovine mammary explants from a lactating cow were incubated in medium containing insulin at 1 μg mL⁻¹, with or without estrogen at 200 ng mL⁻¹. The secretion of cholesterol to the medium in which explants were incubated with insulin was 14.5  μg mL⁻¹ per milligram of tissue; addition of estrogen to the medium containing insulin elevated the cholesterol secretion to level of 20 μg mL⁻¹ per milligram of tissue. Results are least squares means ± SE (n = 4). Bars with different letters differ at P < .05. (b) Effects of prolactin, leptin, and leptin antagonist on cholesterol secretion to the medium from mammary explants culture. Bovine mammary explants from a lactating cow were incubated in medium with or without prolactin at 1 μg mL⁻¹, with or without leptin at 100 ng mL⁻¹, or with a combination of leptin at 100 ng mL⁻¹ and prolactin at 1 μg mL⁻¹, with or without leptin antagonist. Addition of leptin to the medium did not affect the secretion of cholesterol to the medium. Prolactin at a concentration of 1 μg mL⁻¹ elevated the secretion of cholesterol 16 μg mL⁻¹ per milligram of tissue. A combination of leptin at 100 ng mL⁻¹ and prolactin at 1 μg mL⁻¹ elevated the secretion of cholesterol to its highest level of 19 μg mL⁻¹ per milligram of tissue. Addition of leptin antagonist to the leptin plus prolactin combined treatment down-regulated the secretion of cholesterol to 13.2 μg mL⁻¹ per milligram of tissue. Results are least squares means ± SE (n = 3). Bars with different letters differ at P < .05.