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Review
, 9 (1), 204

Key Stages in Mammary Gland Development. Secretory Activation in the Mammary Gland: It's Not Just About Milk Protein Synthesis!

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Review

Key Stages in Mammary Gland Development. Secretory Activation in the Mammary Gland: It's Not Just About Milk Protein Synthesis!

Steven M Anderson et al. Breast Cancer Res.

Abstract

The transition from pregnancy to lactation is a critical event in the survival of the newborn since all the nutrient requirements of the infant are provided by milk. While milk contains numerous components, including proteins, that aid in maintaining the health of the infant, lactose and milk fat represent the critical energy providing elements of milk. Much of the research to date on mammary epithelial differentiation has focused upon expression of milk protein genes, providing a somewhat distorted view of alveolar differentiation and secretory activation. While expression of milk protein genes increases during pregnancy and at secretory activation, the genes whose expression is more tightly regulated at this transition are those that regulate lipid biosynthesis. The sterol regulatory element binding protein (SREBP) family of transcription factors is recognized as regulating fatty acid and cholesterol biosynthesis. We propose that SREBP1 is a critical regulator of secretory activation with regard to lipid biosynthesis, in a manner that responds to diet, and that the serine/threonine protein kinase Akt influences this process, resulting in a highly efficient lipid synthetic organ that is able to support the nutritional needs of the newborn.

Figures

Figure 1
Figure 1
Histological features of the mammary gland of FVB mice during pregnancy and lactation. Mammary glands were isolated from FVB mice on (a,b) day 6 (P6), (c,d) day 12 (P12), and (e,f) day 18 (P18) of pregnancy, and (g,h) day 2 (L2) and (i,j) day 9 (L9) of lactation, fixed in neutral-buffered formalin, sectioned and stained with hematoxylin and eosin. Scale bars in (a, c, e, g and i) represent 100 μm, while those in (b, d, f, h and j) represent 10 μm.
Figure 2
Figure 2
The size and location of cytoplasmic lipid droplets (CLDs) changes upon secretory activation. Mammary glands were isolated from FVB mice on pregnancy (P) days (a) 12, (b) 16, and (c) 18, and (d) day 2 of lactation (L2). Tissues were fixed in neutral-buffered formalin, stained with anti-adipophilin (ADRP) antibody and Alexa Fluor 594 conjugated secondary antibody to outline the cytoplasmic lipid droplets (appearing in red), Alexa Fluor 488-conjugated wheat germ agglutinin to outline the luminal surface of the luminal space of the secretory alveoli (appearing in green), and 4',6-diamino-2-phenylindole (DAPI) to stain the nuclei of mammary epithelial cells (appearing in blue). Idealized schematic drawings, not meant to represent the micrographs shown in the top panel, illustrate the positions of the luminal space (labeled LU), nuclei (purple), and CLDs (labeled red) at pregnancy days (e) 12, (f) 16, and (g) 18, and (h) day 2 of lactation. The scale bars in (a-d) represent 10 μm. Luminal space is indicated by the letters 'Lu', and the white arrowheads indicate CLDs.
Figure 3
Figure 3
Summary of gene expression during pregnancy and lactation by functional class. Adipocyte specific genes decline throughout pregnancy and early lactation while milk protein genes as a class increase over the same time period. The expression of other classes is stable during pregnancy, possibly representing expression in both the adipose and epithelial compartment and increases two- to three-fold (fatty acid and cholesterol synthesis) or decreases about two-fold (fatty acid and protein degradation) at parturition. Adipocyte genes, red; β-oxidation genes, navy blue; proteosome genes, teal; milk protein genes, brown; fatty acid biosynthesis genes, light brown; cholesterol biosynthetic genes, pink.
Figure 4
Figure 4
Expression patterns of milk protein genes. The main graph shows genes whose expression increases more than two-fold at parturition. The inset shows genes with casein-like expression patterns whose mRNA increases mainly during pregnancy. All data are normalized to the level of expression at day 17 of pregnancy (P17). ADPH, adipophilin; MFGM, milk fat globule-EGF-factor; PTHrP, parathyroid hormone related protein; WAP, whey acidic protein; WDNM1, Westmeade DMBA8 nonmetastatic cDNA1; xanthine DH, xanthine oxidoreductase.
Figure 5
Figure 5
Regulation of glucose entry and utilization in the lactating mammary alveolar cell. (a) Glucose enters the cell via glucose transporter (GLUT)1, a non-insulin sensitive transporter. Free glucose enters the Golgi via GLUT1 where it is combined with UDP-galactose, also derived from glucose to make lactose. Since the Golgi membrane is not permeable to disaccharides, lactose draws water osmotically into the Golgi compartment. Glucose is also converted to glucose-6-PO4 by hexokinase. The glucose-6-PO4 can be isomerized by glucose-6-PO4 isomerase to fructose-6-PO4 from whence it is made into pyruvate or glycerol-3-PO4. Glucose-6-PO4 may also enter the pentose phosphate shunt, a major source of NADPH for lipid synthesis. Pyruvate enters the mitochondrion where two major products are ATP, which provides energy to synthetic processes in the cell, and citrate. Citrate has two fates: it serves as the substrate for fatty acid synthesis by conversion to malonyl-CoA and it can be converted to pyruvate through the malate shunt, which provides additional NADPH. NADPH, glycerol-3-PO4, and pyruvate all contribute to triglyceride (TAG) synthesis. (b) Profile of GLUT1, citrate synthase, the citrate transporter, ATP citrate lyase, and glucose phosphate isomerase showing upregulation of the first four and down regulation of the last. (c) Profile of enzymes whose mammary expression is downregulated by a high fat diet. (d) Profile of enzymes that lead to synthesis of polyunsaturated, long chain fatty acids in the mouse mammary gland. P17, day 17 of pregnancy.
Figure 6
Figure 6
Sources of substrate for milk lipid synthesis. The substrate for triacylglycerol synthesis depends on plasma sources of substrate. In the high fat fed animal, such as the usual lactating women who consumes up to 40% of her calories as lipid, fatty acids and glycerol for the synthesis of milk triglycerides (TAGs) originate in the chylomicra and very low density lipoprotein (VLDL) of the liver, whereas only about 10% of TAGs are derived from glucose. During a fasting state, fatty acids continue to be derived from the plasma, but now are transported to the mammary gland directly from the adipose tissue bound to albumin or indirectly as VLDL derived from the liver. In the animal fed a low fat diet, such as the laboratory mouse on the usual chow, a much larger proportion of the fatty acids for TAG synthesis are derived from glucose via the fatty acid synthetic pathways shown in Figure 4. BM, basement membrane; DHAP, dihydroxyacetone phosphate; ER, endoplasmic reticulum; FA, fatty acid; FABP, fatty acid binding protein; GLUT, glucose transporter; LPL, lipoprotein lipase.
Figure 7
Figure 7
Expression of regulatory genes during secretory differentiation and activation. Dotted lines show genes that decrease at least ten-fold during pregnancy, consistent with adipocyte localization. The solid lines show genes that increase at least two-fold at the onset of lactation with much smaller changes during pregnancy. These genes are likely to be important in initiating metabolic changes at secretory activation. LXR, liver X receptor; P17, day 17 of pregnancy; PPAR, proliferator-activated receptor; PrlR, prolactin receptor; SREBP, sterol regulatory element binding protein.
Figure 8
Figure 8
Model predicting critical regulators of secretory activation in the mammary gland. The transcription of milk protein genes is induced by the binding of prolactin to its receptor (the PRLR) and regulated by the STAT5 and ELF5 transcription factors. Translation of milk protein genes may be enhanced by Akt1 acting on their substrates, such as glycogen synthase kinse (GSK)-3/eIF2B, mammalian target of rapamycin (mTOR)/S6 kinase and mTOR/4E-BP1. Transcription of glucose transporter (GLUT)1 may be induced by the PRLR and Akt1 may contribute to either the expression or localization of GLUT1. The response of the mammary gland to dietary fat is sensed by sterol regulatory element binding protein (SREBP), and the stability of SREBP may be enhanced by Akt1-mediated inhibition of GSK3, since phosphorylation of SREBP by GSK3 enhances the ubiquitination and degradation of SREBP in the nucleus.

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