Adipocytes Are the Only Site of Glutamine Synthetase Expression Within the Lactating Mouse Mammary Gland

Abstract

Background: Glutamine in milk is believed to play an important role in neonatal intestinal maturation and immune function. For lactating mothers, glutamine utilization is increased to meet the demands of the enlarged intestine and milk production. However, the source of such glutamine during lactation has not been studied.

Objectives: We aimed to assess the effects of lactation on the expression of glutamine synthetase (GS) in the mammary gland and other tissues of lactating mice.

Methods: Mouse tissues were sampled at 4 time points: 8-wk-old (virgin, control), post-delivery day 5 (PD5, early lactation), PD15 (peak lactation), and involution (4 days after weaning at PD21). We examined the gene expression and protein concentrations of GS and the first 2 enzymes of branched-chain amino acid catabolism: branched-chain aminotransferase 2 (BCAT2) and branched-chain ketoacid dehydrogenase subunit E1α (BCKDHA).

Results: The messenger RNA (mRNA) expression and protein concentrations of GS in mammary glands were significantly lower at PD5 and PD15 compared with the control but were restored at involution. Within the mammary gland, GS protein was only detected in adipocytes with no evidence of presence in mammary epithelial cells. Compared with the control, mRNA and protein concentrations of BCAT2 and BCKDHA in mammary glands significantly decreased during lactation and involution. No changes in GS protein concentrations during lactation were found in the liver, skeletal muscle, and lung. In non-mammary adipose tissue, GS protein abundance was higher during lactation compared with the virgin.

Conclusions: This work shows that, within the mouse mammary gland, GS is only expressed in adipocytes and that the relative GS abundance in mammary gland sections is lower during lactation. This suggests that mammary adipocytes may be a site of glutamine synthesis in the lactating mouse. Identifying the sources of glutamine production during lactation is important for optimizing milk glutamine concentration to enhance neonatal and maternal health.

Keywords: BCAA; adipocytes; glutamine; glutamine synthetase; lactation; mammary gland; mouse.

FIGURE 1

Schematic of study design. Tissues were collected from female mice at 4 time points: virgin (8-wk-old non-lactating virgin mice), post-delivery day 5 (PD5, early lactation), post-delivery day 15 (PD15, peak lactation), and 4 d after weaning at day 21 (involution [INV]). The figure was created with BioRender.

FIGURE 2

Glutamine synthetase is lower in the mammary gland during lactation. (A) mRNA expression and (B) relative protein abundance of GS in the mammary gland at 4 different time points. For Western blotting 10 μg of protein was loaded in each lane. Total protein stained by Coomassie Blue was used for loading control. A representative immunoblot is shown. Full immunoblots with total protein stained with Coomassie Blue corresponding to this figure are in Supplemental Figure 1. Relative GS abundance was normalized to total protein and expressed as fold of virgin group. ∗P < 0.05. Data are presented as mean ± SEM with each dot representing a data point. GS, glutamine synthetase; INV, involution; PD5, post-delivery day 5; PD15, post-delivery day 15.

FIGURE 3

Glutamine synthetase protein is localized in mammary adipocytes. Immunohistochemistry staining of GS in the mammary gland at 4 time points. Arrows and an arrowhead point at the brown staining of GS. Nuclei are stained blue with 4′,6-diamidino-2-phenylindole (DAPI). The arrowhead indicates cells that were also stained for UCP1 in virgin samples. Clusters of blue nuclei surrounding alveoli present at PD5 and PD15 indicate mammary epithelial cells. Representative images are shown. Scale bars = 75 μm. GS, glutamine synthetase; INV, involution; PD5, post-delivery day 5; PD15, post-delivery day 15; UCP1, uncoupling protein 1.

FIGURE 4

Branched-chain aminotransferase 2 is lower in the mammary gland during lactation. (A) mRNA expression and (B) relative protein abundance of BCAT2 in the mammary gland at 4 different time points. For Western blotting 25 μg of protein were loaded in each lane. Total protein stained by Coomassie Blue was used for loading control. A representative immunoblot is shown. Full immunoblots with total protein stained with Coomassie Blue corresponding to this figure are in Supplemental Figure 3. Relative BCAT2 abundance was normalized to total protein and expressed as a fold of virgin group. ∗P < 0.05. Data are presented as mean ± SEM with individual data points shown as dots. BCAT2, branched-chain aminotransferase 2, INV, involution; PD5, post-delivery day 5; PD15, post-delivery day 15.

FIGURE 5

Branched-chain ketoacid dehydrogenase is lower in mammary gland during lactation. (A) mRNA expression and (B) relative protein abundance of BCKDHA in the mammary gland at 4 different time points. For Western blotting 30 μg of protein were loaded in each lane. Total protein stained by Coomassie Blue was used for loading control. A representative immunoblot is shown. Full immunoblots with total protein stained with Coomassie Blue corresponding to this figure are in Supplemental Figure 4. Relative BCKDHA abundance was normalized to total protein and expressed as a fold of virgin group. ∗P < 0.05. Data are presented as mean ± SEM with individual data points shown as dots. BCKDHA, branched-chain ketoacid dehydrogenase subunit E1α; INV, involution; PD5, post-delivery day 5; PD15, post-delivery day 15.

FIGURE 6

Glutamine synthetase protein abundance in retroperitoneal adipose tissue during lactation. Relative protein abundance of GS in retroperitoneal adipose tissue at 4 different time points. Ten micrograms of protein was loaded in each lane. Total protein stained by Coomassie Blue was used for loading control. A representative immunoblot is shown. Full immunoblots with total protein stained with Coomassie Blue corresponding to this figure are in Supplemental Figure 6. Relative GS abundance was normalized to total protein and expressed as a fold of virgin group. ∗P < 0.05. Data are presented as mean ± SEM with individual data points shown as dots. GS, glutamine synthetase, INV, involution; PD5, post-delivery day 5; PD15, post-delivery day 15.