Targeting of GLUT1 Glucose Transporter
Larry H. Bernstein, MD, FCAP, Curator
LPBI
Prolactin-induced Subcellular Targeting of GLUT1 Glucose Transporter in Living Mammary Epithelial Cells
Arieh Riskin, M.D., M.H.A., and Yehudit Mond, M.Sc.
Rambam Maimonides Med J Oct 2015; 6( 4): e003 http://www.rmmj.org.il/userimages/525/1/PublishFiles/531Article.pdf
Abbreviations: ECFP, enhanced cyan fluorescent protein; EGFP, enhanced GFP; GFP, green fluorescent protein; GM, growth medium; HMEC, human mammary epithelial cells; MEC, mammary epithelial cells; MMEC, mouse mammary epithelial cells; pECFP, plasmid vector ECFP; pEGFP, plasmid vector EGFP; PCR, polymerase chain reaction; SM, secretion medium. Citation: Riskin A, Mond Y. Prolactin-induced Subcellular Targeting of GLUT1 Glucose Transporter in Living Mammary Epithelial Cells. Rambam Maimonides Med J 2015;6 (4):e0038. http://dx.doi.org:/10.5041/RMMJ.10223
Background: Studying the biological pathways involved in mammalian milk production during lactation could have many clinical implications. The mammary gland is unique in its requirement for transport of free glucose into the cell for the synthesis of lactose, the primary carbohydrate in milk. Objective: To study GLUT1 trafficking and subcellular targeting in living mammary epithelial cells (MEC) in culture.
Methods:Immunocytochemistry was used to study GLUT1 hormonally regulated subcellular targeting in human MEC (HMEC). To study GLUT1 targeting and recycling in living mouse MEC (MMEC) in culture, we constructed fusion proteins of GLUT1 and green fluorescent protein (GFP) and expressed them in CIT3 MMEC. Cells were maintained in growth medium (GM), or exposed to secretion medium (SM), containing prolactin.
Results: GLUT1 in HMEC localized primarily to the plasma membrane in GM. After exposure to prolactin for 4 days, GLUT1 was targeted intracellularly and demonstrated a perinuclear distribution, co-localizing with lactose synthetase. The dynamic trafficking of GFP-GLUT1 fusion proteins in CIT3 MMEC suggested a basal constitutive GLUT1 recycling pathway between an intracellular pool and the cell surface that targets most GLUT1 to the plasma membrane in GM. Upon exposure to prolactin in SM, GLUT1 was specifically targeted intracellularly within 90–110 minutes.
Conclusions: Our studies suggest intracellular targeting of GLUT1 to the central vesicular transport system upon exposure to prolactin. The existence of a dynamic prolactin-induced sorting machinery for GLUT1 could be important for transport of free glucose into the Golgi for lactose synthesis during lactation.
KEY WORDS: CIT3 mouse mammary epithelial cells, green fluorescent protein, GLUT1 glucose transporter, human mammary epithelial cells, prolactin
Biology of Milk Production and Transport Pathways of Milk Constituents Females of all mammalians bear mammary glands, and milk secretion and lactation is a characteristic feature of all mammalian species, which are the only organisms that produce copious glandular skin secretions to feed their young.1,2 Lactation is a highly complex and evolutionarily ancient strategy of all mammals, providing their offspring with a highly digestible, concentrated, nutritionally balanced diet, while allowing adult mammals to evolve a wide range of developmental and reproductive strategies and specialize on diets that could either be too difficult to capture or digest or would be insufficient to cover the high nutritional needs of their small rapidly growing offspring.1–3 Lactation helps mammalian mothers cope with unreliable food supplies, because lactating females can draw on their nutrient reserves for milk production, suggesting an evolutionary advantage for their dependent offspring, since milk intake promotes growth, fitness, and survival of the young.2,4,5 Beyond nourishment of the neonate, milk also helps establish immunological and endocrine competence in the offspring. Milk’s nutrient composition varies extensively across mammalian species, as a function of evolutionary history, maternal nutrient intake, duration of milk production, and stage of lactation.2,5 Milk is a complex mixture whose composition reflects different transport and secretion mechanisms within the mammary gland that aim to answer the different nutritional needs of mammalian neonates.6
The lactating mammary gland is composed of branching ducts ending in alveolar clusters where milk is produced. A single layer of polarized secretory epithelial cells forms the alveolar wall. The alveoli are surrounded by myoepithelial cells and are embedded in vascularized connective tissue stroma. While growth of the mammary gland and secretion of milk are stimulated by growth hormone, prolactin, adrenocortical steroids, estrogens, and progesterone, ejection of milk requires contraction of myoepithelial cells stimulated by oxytocin.6,7 The cytoplasm of the secretory alveolar epithelial cells is filled with numerous mitochondria, extensive rough endoplasmic reticulum network, well-developed Golgi apparatus, and secretory vesicles in the apical region of the cell adjacent to the alveolar lumen. The basal side of the alveolar epithelial cells lies on a basement membrane that separates them from the stroma and vascular system. In between, epithelial cells are connected to each other by an apical complex of tight junctions that inhibit direct paracellular exchange of substances between the vascular compartment and milk in the alveolar lumen. There are five pathways by which solutes, including proteins, lipids, ions, nutrients, and water, can be transported into the milk. Four are transcellular, involving transport across at least two membrane barriers, while the fifth is para-cellular and allows direct exchange of interstitial and milk components: (1) The exocytic pathway is for endogenously generated aqueous soluble substances, including lactose, oligosaccharides, the major milk proteins, citrate, phosphate, calcium, and other nutrients, which is similar to exocytic pathways in other cell types; (2) The transport pathway for milk lipids is unique to the mammary gland generating the milk fat globules; milk lipids, primarily triacylglycerides, are synthesized in the smooth endoplasmic reticulum in the basal region of the lactating alveolar cell—the newly synthesized lipid molecules are coated by protein membranes to form small storage structures called cytoplasmic lipid droplets that are transported to the apical plasma membrane, where they are secreted by a unique budding process as membrane-enveloped structures, i.e. the milk fat globules; (3) The trans-cytosis pathway transports macromolecules derived from the serum or stromal cells, including serum proteins (such as immunoglobulin, albumin, and transferrin), hormones (such as insulin, prolactin, and estrogen), and stromalderived substances (such as secretory immunoglobulin A, cytokines, and lipoprotein lipase); (4) Various membrane transport pathways transport ions and small molecules (such as glucose, amino acids, and water) across the basal and apical plasma membranes of the polarized alveolar epithelial cell; and (5) The para-cellular pathway provides a direct route for transfer of serum and interstitial substances into the milk. These transport pathways are regulated by the functional stage of the mammary gland and by hormones and growth factors.6
Studying mammary gland biology, transport pathways of milk constituents, and their influence on milk production in general, and specifically investigating glucose transport mechanisms in mammary epithelial cells (MEC) that could influence lactose synthesis and thus milk volume, is important and could have many clinical implications, including in breastfeeding support to mothers, in the dairy industry, and in oncology. Breast cancer tumor cells may utilize some of the MEC transport pathways to support metabolically their rapid uncontrolled growth.6,8
Glucose Transport Pathways in the Mammary Epithelial Cells Human milk is rich in lactose, which is the major osmotic constituent of human milk and thus the major determinant of milk volume.9 Lactose is a disaccharide composed of glucose and galactose. Lactose is found only in milk and is the primary carbohydrate in milk. The final step in the biosynthesis of lactose from UDP-galactose and glucose is catalyzed by lactose synthetase, a complex of - lactalbumin and the Golgi enzyme 1,4-galactosyltransferase.10 1,4-Galactosyltransferase is embedded in the inner surface of Golgi membranes. It is membrane-bound and directed towards the lumen of the cisternal space.10,11 Galactosyltransferase is found in most tissues and is involved in protein glycosylation. In mammals 1,4-galactosyltransferase has been recruited for a second biosynthetic function, the production of lactose. This function takes place exclusively in the lactating mammary gland. Galactosyltransferase has a relatively poor affinity for glucose (Km~1 M). The affinity of this enzyme is profoundly modified by transient association with -lactalbumin, which creates a binding site for glucose, so that the affinity of the transferase for glucose increases about 500-fold (Km~2 mM).10 This allows the synthesis of lactose in the Golgi to occur at the physiological concentrations found in the mammary cell. -Lactalbumin is a milk whey protein that is not catalytically active by itself, but is necessary for the synthesis of lactose. The initiation of -lactalbumin synthesis that occurs at parturition is required for the initiation of copious milk production, but is neither the only factor nor the limiting factor controlling lactose synthesis.11,12 Availability of glucose and UDP-galactose to the lactose synthase enzyme complex in the Golgi apparatus may be rate-limiting for lactose synthesis. Many cells possess active mechanisms for the uptake of nucleotide sugars such as UDP-galactose into the Golgi, which are essential to protein glycosylation. In contrast, the mammary gland is unique in its need to transport free glucose into the Golgi. The main known isoform of glucose transporters expressed in the mammary gland is GLUT1 (SLC2A1).9,13,14–20 Levels of GLUT1 increase progressively during pregnancy, reaching their highest levels during lactation,15,16 and this is dependent on prolactin.21 In most cells GLUT1 normally resides in the plasma membrane and is responsible for basal glucose uptake. In polarized epithelial cells, including mammary cells, GLUT1 is targeted primarily to the basolateral membrane.22 The role of GLUT1 in glucose transport into Golgi has been controversial.15,23,24 Evidence from in vivo and in vitro studies demonstrated unique hormonally regulated intracellular targeting of GLUT1 from the plasma membrane to a low-density intracellular compartment in mouse mammary gland during lactation.16,23–25 Further work distinguished this compartment from Golgi, suggesting that the hormonally induced intracellular targeting of GLUT1 in lactating MEC is into a Brefeldin A-sensitive low-density vesicle that may represent a subcompartment of cisGolgi.25 This hormonally regulated subcellular targeting of GLUT1 may have an important role for lactose synthesis in MEC during lactation.
The aim of this work was to study GLUT1 subcellular targeting in living human and mouse mammary epithelial cells (HMEC and MMEC) in culture; and to study GLUT1 intracellular trafficking in living MMEC in culture under the effects of the lactogenic hormones that regulate it. Our hypothesis was that there would be dynamic basal trafficking of GLUT1 in living MEC that would target most GLUT1 to the basolateral membrane under maintenance conditions, and intracellularly upon exposure to prolactin, which should be the main hormonal stimulus that drives this translocation during lactation. Our studies could then complete the previous in vitro findings in fixed cell25 by adding the dynamic observations in living MEC that could in turn relate to the in vivo findings.16
RESULTS
GLUT1 Subcellular Targeting in HMEC Immunocytochemistry of HMEC in maintenance MEGM medium using highly specific anti-GLUT1 antibody demonstrated plasma membrane distribution of GLUT1 as well as an intracellular, mostly perinuclear, pattern (Figure 1A). After exposure to the prolactin-rich MESM for 4 days, GLUT1 was specifically targeted intracellularly, demonstrating a perinuclear pattern. A distinct nuclear membrane distribution of GLUT1 was also observed under these conditions (Figure 1B). In secretion medium GLUT1 green signal colocalized with the blue signal of pECFP-Golgi (Figure 2A, B, C). It also co-localized with the red signal of alpha-lactalbumin and alpha-mannosidase II (Figure 2D, E, F, and G, H, I, respectively). Partial co-localization was demonstrated with the red signal of beta-COP and with transferrin-Texas Red (Figure 3A, B, C, and D, E, F, respectively). No co-localization was demonstrated after staining the cells with the red stain, BODIPY-TR ceramide (Figure 4A, B, C, D, E, F). GLUT1 Fusion Chimeras to EGFP Exhibit Normal GLUT1 Targeting in Vitro We subcloned GLUT1 cDNA into pEGFP to create N- and C-terminus fusions. The recombinant plasmid vectors were introduced into CIT3 cells by transient liposome-mediated transfection, achieving fluorescent expression in 20%–30% of the cells.
Figure 1. Exposure to Prolactin Causes Intracellular Targeting of GLUT1. Cells were fixed and exposed to specific anti-GLUT1 primary antibody. Bar 15 m. A: In maintenance medium, GLUT1 demonstrates primarily a plasma membrane distribution as well as some intracellular mostly perinuclear staining. B: After exposure to prolactin-rich medium for 4 days, GLUT1 was specifically targeted intracellularly, demonstrating a perinuclear pattern, as well as a distinct nuclear membrane staining.
Figure 2. After Exposure to Prolactin, GLUT1 Colocalizes with ECFP-Golgi, alpha-Lactalbumin and alpha- Mannosidase II. Fluorescent images were captured 60 hours after transfection with 1 microgram of pECFP-Golgi. Cells were maintained in prolactin-rich medium for 4 days, before they were fixed and stained with specific anti-GLUT1, anti-alpha- lactalbumin or anti-alpha-mannosidase II. GLUT1 is shown in green, and alpha-lactalbumin or alpha-mannosidase II in red after staining with FITC-conjugated and Texas Redconjugated secondary antibodies, respectively. ECFPGolgi emits cyan-blue fluorescence when exposed to fluorescent light at the appropriate wavelength. Bar 10 microm. A, D, G: GLUT1 signal. B: ECFP-Golgi signal. E, H: alpha-Lactalbumin and alpha-mannosidase II signals, respectively. C, F, I: Superimposed images. Perinuclear colocalization of GLUT1 and ECFP-Golgi is shown as areas of coincident staining (C). Co-localization of GLUT1 and alpha-lactalbumin or alpha-mannosidase II appear as areas of coincident staining, giving rise to yellow signal (F, I).
Figure 3. After Exposure to Prolactin, GLUT1 only Partially Co-localizes with beta-COP and TransferrinTexas Red in Endosomes. Cells were maintained in prolactin-rich medium for 4 days, before they were fixed and stained with specific anti-GLUT1 or anti-beta-COP primary antibodies. Some cells were exposed shortly to transferrin-Texas Red staining before fixation and exposure to anti-GLUT1. GLUT1 is shown in green, and beta-COP in red after staining with FITC-conjugated and Texas Redconjugated secondary antibodies, respectively. Transferrin stain appears in red. A, D: GLUT1 signal. B, E: beta-COP and transferrin (short-term exposure) signals, respectively. C, F: Superimposed images. Partial co-localization of GLUT1 with beta-COP or transferrin in endosomes appears as areas of coincident staining, giving rise to yellow signal.
Figure 4. After Exposure to Prolactin, GLUT1 Does not Co-localize with BODIPY-TR Ceramide. Cells were maintained in prolactin-rich medium for 4 days, before they were fixed and stained with specific anti-GLUT1 primary antibody, or stained with BODIPYTR ceramide. GLUT1 is shown in green after staining with FITC-conjugated secondary antibody. BODIPY-TR ceramide appears in red. A, D: GLUT1 signal. B, E: BODIPY-TR ceramide signal. C, F: Superimposed image. There is little overlap of GLUT1 green signal with BODIPY-TR ceramide.
GLUT1 Fusion Chimeras to EGFP Exhibit Normal GLUT1 Targeting in Vitro We subcloned GLUT1 cDNA into pEGFP to create N- and C-terminus fusions. The recombinant plasmid vectors were introduced into CIT3 cells by transient liposome-mediated transfection, achieving fluorescent expression in 20%–30% of the cells.
Immunocytochemistry studies with antibodies against the C-terminus of native GLUT1 showed that the fluorescent signal of both GLUT1 chimeras to GFP co-localized extensively with native GLUT1 (Figure 5). This result validated the use of the GLUT1-GFP fusion proteins to study dynamic aspects of GLUT1 targeting.
Figure 5. GLUT1 Chimeras to GFP Co-localize with Native GLUT1 in CIT3 Cells in SM. EGFP-GLUT1 fusion protein (B) exhibits the same intracellular distribution as native GLUT1 (A). Superimposed images (C) demonstrate that co-localization of native GLUT1 and EGFP-GLUT1 fusion protein appears as areas of coincident staining, giving rise to yellow signal. GLUT1-EGFP fusion protein (E) exhibits the same intracellular distribution as native GLUT1 (D). Superimposed images (F) demonstrate that co-localization of native GLUT1 and GLUT1-EGFP fusion protein appears as areas of coincident staining, giving rise to yellow signal.
EGFP chimeras to GLUT1 demonstrated change in subcellular distribution, after 96 hours of exposure to SM. In GM the fusion proteins were targeted mainly to the basolateral plasma membrane. In SM GLUT1-EGFP chimeras were mostly targeted into the cell, exhibiting unique perinuclear distribution with punctate pattern scattered through the cytoplasm (Figures 5 and 6). Both the N- and C-terminus fusions to GLUT1 (EGFP-GLUT1 and GLUT1-EGFP, respectively) exhibited the same targeting patterns (Figure 6).
Figure 6. CIT3 Cells Expressing EGFP Fusion to the N- and C-termini of GLUT1 in GM and after 4 Days in SM. High-power images. Upper left: Plasma membrane targeting of EGFP-GLUT1 in GM. Lower left: Intracellular pattern of EGFP-GLUT1 signal in differentiated cells in SM. Upper right: Plasma membrane targeting of GLUT1-EGFP in GM. Lower right: Intracellular targeting of GLUT1-EGFP with perinuclear punctate distribution in cells exposed to SM. All images captured with 0.25 s exposure time and are at 40× magnification, except for the upper left image, which was optimized at 0.5 s exposure time and higher power (60×).
Degree of Differentiation Affects Intracellular Targeting of GLUT1 Static images of cells at different levels of differentiation in SM demonstrated that the degree of differentiation affected the level and distribution of GLUT1 targeting in perinuclear localization (Figure 7). In GM the GLUT1-EGFP signal was mostly targeted to the plasma membrane (Figure 7A). In SM GLUT1-EGFP fusion proteins exhibited the unique perinuclear punctate pattern, described above (Figure 7B). However, in the more differentiated MEC in SM, where vesicles and fat globules were morphologically prominent, the GLUT1-EGFP signal was no longer punctate, but targeted to the boundaries of these vesicles, still maintaining mostly perinuclear distribution. There was signal throughout the cytoplasm as well (Figure 7C, D). This differentiation-related GLUT1 subcellular targeting may also support the assumption that dynamic transport systems are involved in the trafficking of the GFP-GLUT1 fusion proteins, as will be shown below.
Figure 7. Degree of Differentiation Affects Intracellular Targeting of GLUT1. Static images of CIT3 cells in GM and at different levels of differentiation in SM. Panel A: In GM; B, C, D: In SM at different levels of differentiation. The upper left figure of each panel is GLUT1-EGFP signal. The upper right figure is staining of the same cells with BODIPY-TR ceramide (Molecular Probes, Inc., Eugene, OR, USA), which is a dye that marks the trans-Golgi in red. Living CIT3 cells were pre-incubated with 5 nmol/mL of BODIPY-TR ceramide at 4C for 30 minutes. Lower left figure is superimposed image of the upper figures. Lower right figure is phase contrast image of the cells to show the different levels of differentiation.
GLUT1 Intracellular Trafficking in Secretion Media Seems to be Dynamic Living CIT3 cells transfected with GLUT1-EGFP and kept in SM were followed by time-lapse imaging, where trafficking of GLUT1 fusion proteins could be seen (Figure 8; also available online as a supplemental YouTube video clip). This trafficking seems to be dynamic and could suggest that transport systems are possibly involved in it.
Figure 8. GLUT1 Intracellular Trafficking in SM is Dynamic. Figure 8 is also available online as a YouTube video clip at: https://youtu.be/lEby2cqSDek. The figure plates are from frames 8 minutes apart. All images captured with 0.25 s exposure time and are at 40× magnification.
Secretion Medium Induces GLUT1 Intracellular Targeting in Living Mammary Cells When living CIT3 cells, transfected with GLUT1- EGFP and kept in GM, were exposed to SM, dynamic trafficking of GLUT1 fusion proteins was demonstrated intracellularly, starting after approximately 50–60 minutes, with maximal intracellular targeting within 90–110 minutes. When the cells were returned to GM, most of the changes were reversible within 1–2 hours, although not fully, with redistribution of the fluorescent GLUT1 chimera mostly in the plasma membrane (Figure 9; also available online as a supplemental YouTube video clip).
Figure 9. Lactogenic Hormones in SM Induce GLUT1 Intracellular Targeting in Living Mammary Cells. Figure 9 is also available online as a YouTube video clip at: https://youtu.be/3oYv21jcAj4. The figure plates are from frames 16 minutes apart. All images captured with 0.5 s exposure time and are at 40× magnification.
Prolactin Induces GLUT1 Intracellular Targeting in Living Mammary Cells Exposure of CIT3 cells kept in GM to SM containing prolactin without hydrocortisone caused the same changes in GLUT1 subcellular targeting as were seen with full SM. Dynamic trafficking of GLUT1 fusion proteins intracellularly was demonstrated, starting after approximately 50–60 minutes, with maximal intracellular targeting within 90–110 minutes. When the cells were returned to GM, most of the changes were reversible within 1–2 hours, although not fully. The same response was reproduced with different prolactin concentrations (300 ng/mL, 30 ng/mL), as low as 3 ng/mL (compared to the 3 microg/mL of prolactin usually used in SM). Representative results after exposure of CIT3 cells kept in GM to 30 ng/mL prolactin are shown (Figure 10; also available online as a supplemental YouTube video clip). We were not able to demonstrate doseresponse relations with the different prolactin concentrations, possibly because the results are qualitative, rather than quantitative. There was no difference in the time required to achieve maximal effect with different prolactin concentrations. Secretion medium containing hydrocortisone (3 microg/mL as in full SM) without any prolactin caused no change in GLUT1 subcellular distribution.
Figure 10. Prolactin Induces GLUT1 Intracellular Targeting in Living Mammary Cells. The response here was reproduced with prolactin concentrations of 30 ng/mL. Figure 10 is also available online as a YouTube video clip at: https://youtu.be/bUkUHZ0soPI. The figure plates are from frames 20 minutes apart. All images captured with 2 s exposure time and are at 60× magnification.
DISCUSSION Human milk contains about three times more lactose than does rodent milk, suggesting that glucose transport mechanisms can be very important in humans. Thus, before studying GLUT1 subcellular trafficking in a CIT3 MMEC model, we first examined whether hormonally regulated subcellular targeting of GLUT1 occurs in HMEC upon conditions mimicking lactation. For this, we utilized immunofluorescent staining of HMEC to demonstrate prolactin-dependent co-localization of GLUT1 with several Golgi markers.
The distribution of GLUT1, as demonstrated using immunocytochemistry with C-terminusspecific anti-GLUT1 antibody, was primarily in plasma membrane, as well as some intracellular perinuclear punctate pattern, when the cells were maintained in baseline growth medium. Upon exposure to prolactin in secretion media, GLUT1 specifically redistributed from the plasma membrane, demonstrating a perinuclear distribution with a pattern that may represent the Golgi or related structures in the central intracellular vacuolar trafficking system. A distinct perinuclear membrane distribution of GLUT1 was also observed under these conditions. Co-localization studies gave insight as to the possible Golgi subcompartment that GLUT1 resides in. GLUT1 was targeted intracellularly, co-localizing with components of lactose synthetase complex. It co-localized with ECFP-Golgi, the cyan fluorescent protein fused to the membrane-anchoring signal specific to beta 1,4-galactosyltransferase, identifying the medial/trans region of the Golgi. This colocalization gave another spatial support to its role in the transport of free glucose for lactose synthesis by beta 1,4-galactosyltransferase in the inner cisternae of the Golgi apparatus. This was further supported by the co-localization of GLUT1 with alpha-lactalbumin, which is the milk whey protein that is not catalytically active, by itself, but which in association with beta 1,4-galactosyltransferase is necessary for the synthesis of lactose.53 The initiation of alpha-lactalbumin synthesis that occurs at parturition is required for the initiation of copious milk production, but is neither the only factor nor the limiting factor controlling lactose synthesis.54 GLUT1 also colocalized with the medial-Golgi marker, alpha- mannosidase II; however, it did not co-localize with the trans-Golgi marker, BODIPY-TR ceramide. Only partial co-localization was demonstrated with beta- COP, which is a cis-Golgi marker.
The co-localization studies suggest intracellular targeting to the central vesicular transport system, which may represent a cis/medial-Golgi subcompartment. These findings are in agreement with previous findings in the mouse mammary gland16 and in CIT3 MMEC.25,55 Partial co-localization was also noted with transferrin-Texas Red after brief exposure, which marks the endosomes. The possible identification of GLUT1 in endosomes suggests that GLUT1 sorting is a continuous, dynamic process.55 Further work delineating the molecular mechanism of GLUT1 sorting and the targeting determinants it recognizes should improve our understanding of a key regulatory step of milk production in the nursing mother.
Apparent nuclear membrane staining for GLUT1 seen in HMEC has not been reported previously in any cell type, and its significance is a matter for speculation and further study. There is evidence to suggest that the apical nuclear envelope may serve as an intermediary connection between the endoplasmic reticulum and the Golgi.56 Also, the perinuclear staining of GLUT1 noted may actually be one of the perinuclear endosomal recycling compartments.52
Our findings in HMEC illustrate a potential mechanism for the delivery of free glucose to the Golgi, in what may be the rate-limiting step for lactose synthesis and milk production. In addition to its widely recognized role in the uptake of glucose by cells, GLUT1 may also mediate glucose transport between intracellular compartments.
The in vitro study of a dynamic process required developing a system of living cells with labeling of GLUT1. Green fluorescent protein is a reporter molecule for monitoring gene expression and protein localization in vivo, in situ, and in real time.57–63 Green fluorescent protein is expressed in eukaryotic cells as a fusion protein that serves as a “fluorescent tag.” The use of fluorescent fusion proteins of GLUT1 allows the study of the same cells over time, permitting studies of exocytosis and endocytosis, not just steady-state distributions. Using GFP fusion to GLUT1 is ideal for studying intracellular trafficking and subcellular targeting of GLUT1 in MEC under hormonal stimulation. It also permits evaluation of chimeric protein targeting in an antibody-independent fashion and confirms that we are studying GLUT1 and not a novel, lactation specific glucose transporter isoform that shares the GLUT1 epitope. The GLUT1 cDNA sequence29 was subcloned into pEGFP and introduced into CIT3 cells by transient transfection, with over-expression of the fluorescent GLUT1 in approximately a quarter of the cells. The intracellular targeting of GLUT1- EGFP chimera was consistent from cell to cell.
Lactogenic hormones in SM changed subcellular targeting of GFP fusion chimeras to GLUT1 from a plasma membrane distribution to an intracellular pattern, predominantly perinuclear and punctate, but also throughout the cytoplasm. The fact that this pattern was consistent with the distribution of native GLUT1 supported the use of GLUT1 chimeras to GFP as a model for studying GLUT1 intracellular targeting in MEC. Since the behavior and intracellular distribution of both the N- and C-fusion chimeras of GLUT1 to GFP were consistently the same, further studies were carried out with only one of them (GLUT1-EGFP). The level of differentiation of the lactating MEC in SM affected the degree of GLUT1 intracellular targeting and the distribution of its cytoplasmic, mostly perinuclear, localization. This fits well the previous in vivo and in vitro findings16,25 and actually also points to the possible involvement of intracellular membrane vesicular trafficking systems in GLUT1 intracellular targeting.
Living mouse MEC kept in SM demonstrated dynamic trafficking of GLUT1-EGFP fusion proteins. Careful tracking of these fluorescent GLUT1 vesicles excluded random movement and actually suggested that the dynamic intracellular targeting of GLUT1 may be mediated through altering GLUT1 exocytosis and endocytosis.
When CIT3 cells kept in GM were exposed to SM, the changes in GLUT1 targeting from mostly a plasma membrane pattern to an intracellular pattern occurred within 60–120 minutes. The maximal intracellular translocation of GLUT1-EGFP green fluorescent signal after exposure to SM was noted at 100–110 minutes. Some of this effect was reversible within 60–120 minutes upon withdrawal of SM, but we were not able to demonstrate full reversibility of the process in our in vitro system. The relatively rapid changes in GLUT1 targeting in living MMEC exposed to SM, which took place within minutes to hours, were in accordance with the findings from the in vivo studies of forced weaning, demonstrating reversible changes in GLUT1 subcellular targeting within 3–5 hours.16 These findings are also supported by the previous observation that, as early as 15 minutes after exposure of mammary tissue fragments from lactating rabbits to prolactin, the cell morphology already changed with marked increase in the relative volume occupied by the Golgi region.64
The next step was to define which of the hormones in SM is responsible for GLUT1 intracellular targeting. Exposure of CIT3 cells kept in GM to SM containing prolactin without hydrocortisone caused the same changes in GLUT1 subcellular targeting as seen with full SM. The same response was reproduced with prolactin concentrations as low as 3 ng/mL (compared to the 3 g/mL of prolactin usually used in SM). The serum concentration range of prolactin in lactating mothers is 20–300 ng/mL.65 However, we were not able to demonstrate dose-response effects with the different prolactin concentrations. The GLUT1-EGFP intracellular signal translocation took place at approximately the same time (100–120 minutes) with 3 ng/mL prolactin as it did with 3 g/mL of prolactin in the full SM. Further studies are needed to demonstrate dosedependent effects of prolactin, expressed as different levels of intracellular green fluorescent signal of GLUT1 chimeras, but this requires a more quantitative recording of the signal that unfortunately we did not have in these studies. Secretion medium containing hydrocortisone without any prolactin caused no change in GLUT1 subcellular distribution, thus excluding it as a cause for GLUT1 intracellular targeting in SM. Further studies are also needed to explore the effects of prolactin combined with other hormones, such as estrogen.
The suggestion that GLUT1 does not solely act at the plasma membrane, but may function in an intracellular organelle as well, conceptually complements the well-known insulin-regulated targeting of GLUT4,66 and to a lesser extent of GLUT1,38 to their site of action, the plasma membrane, in fat and muscle cells. Our results suggest the existence of a prolactin-induced, cell type-specific, developmental stage-specific sorting machinery for GLUT1 in MEC, and supports glucose transport as a potential ratelimiting step for lactose synthesis during lactation. The ability of the system to respond quickly to hormonal changes by altering the transport, and thus the availability of free glucose for lactose synthesis, is complementary to the well-known insulin-regulated targeting of GLUT4 to the plasma membrane in fat and muscle cells, where GLUT4 is available for glucose uptake into the cell within minutes.67 This machinery offers another level of regulation of lactose synthesis by altering GLUT1 targeting within minutes to hours, as was demonstrated also in vivo. 16 This step may not require new protein synthesis, or increase in the total amount of GLUT1 or enzymes involved in lactose synthesis, which takes longer. Our study relied only on immuno-histochemistry analysis, and further studies including Western blot and real-time PCR are needed to address the possibility of up- or downregulation of GLUT1.
Despite the fact that in our first experiments we have demonstrated specific intracellular targeting of GLUT1 from the plasma membrane of HMEC upon exposure to prolactin, the main limitation of this part of the dynamic studies is that it was limited to MMEC, and more specifically to CIT3 cell lines. Thus, these conclusions cannot be currently generalized or extended to other mammals, including humans. The suggestion that glucose transporters, other than GLUT1, may be involved in glucose regulation in MEC during lactation,18,68–71 and that their role may be more significant in other mammals, and influenced by factors other than lactogenic hormones,72–75 needs to be addressed.
Another limitation of this study is the issue of cell-to-cell variability in the phenotype of mouse MEC in the culture. Cell phenotype could vary in many ways, including morphology, intracellular lipid deposition, apparent states of differentiation, and GLUT1 targeting kinetics. This is an inherent limitation of a descriptive study based on microscopic findings. To decrease a possible selection bias of the cells we were studying, we tried to select representative cells on a lower-power field before studying them in a high-power field. We also repeated each experiment more than three times, and verified that the results were reproducible. Yet, such selection bias cannot be fully excluded.
A further limitation of our descriptive colocalization studies is the use of fluorescent microscopy. Confocal microscopy should be preferred in future studies like this, because it gives higher resolution and better color separation, enables the use of more colors at the same time, and is more accurate in differentiating true colocalization from proteins in close proximity.
Also, being a descriptive morphological study, our study did not deal with the ability of the cells to synthesize and secrete lactose in culture. This is a complex issue that is not dependent only on the presence of lactogenic hormones in the medium, and may be affected by many factors, such as the intracellular matrix. However, if these results were obtained in cells expressing lactose, then the issues of cell-to-cell variations and possible phenotypic effects discussed above would have been minimized.
The fact that this work is based on two previous works, one in vivo on mammary glands from lactating mice16 and the second in vitro on the same CIT3 cells, 25,55 establishes a continuum that supports our results within their limited scope. These results can possibly form the basis and methodological approach for future works in other primary MEC in order to try and generalize the conclusions.
In summary, our results demonstrated a basal constitutive GLUT1 membrane-recycling pathway between an intracellular pool and the cell surface in CIT3 MMEC, which targets most of the GLUT1 to the plasma membrane in GM. As in other cell types it is responsible for maintaining basal glucose uptake. But, in these MEC there is hormonally regulated cell type-specific, developmental stage-specific sorting machinery for GLUT1 intracellular targeting in lactation. This process is induced by prolactin and is highly sensitive to low concentrations of prolactin. It provides the cell with a quick mechanism by which it can supply free glucose intracellularly to serve as substrate for lactose synthesis in the Golgi. This machinery offers another level of regulation of lactose synthesis by altering GLUT1 targeting within minutes to hours, as was demonstrated also in vivo.16 The rapid responsiveness of GLUT1 targeting suggests that this machinery does not require new protein synthesis. It may also support glucose transport as a rate-limiting step for lactose synthesis during lactation.
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The Angelina Jolie Effect in Jewish Law: Prophylactic Mastectomy and Oophorectomy in BRCA Carriers
Background: Following the announcement of actress Angelina Jolie’s prophylactic bilateral mastectomies and subsequent prophylactic oophorectomy, there has been a dramatic increase in interest in BRCA testing and prophylactic surgery.
Objective: To review current medical literature on the benefits of prophylactic mastectomy and oophorectomy among BRCA-positive women and its permissibility under Jewish law.
Results: Recent literature suggests that in BRCA-positive women who undergo prophylactic oophorectomy the risk of dying of breast cancer is reduced by 90%, the risk of dying of ovarian cancer is reduced by 95%, and the risk of dying of any cause is reduced by 77%. The risk of breast cancer is further reduced by prophylactic mastectomy. Prophylactic oophorectomy and prophylactic mastectomy pose several challenges within Jewish law that call into question the permissibility of surgery, including mutilation of a healthy organ, termination of fertility, self-wounding, and castration. A growing number of Jewish legal scholars have found grounds to permit prophylactic surgery among BRCA carriers, with some even obligating prophylactic mastectomy and oophorectomy.
Conclusion: Current data suggest a significant reduction in mortality from prophylactic mastectomy and oophorectomy in BRCA carriers. While mutilation of healthy organs is intrinsically forbidden in Jewish law, the ability to preserve human life may contravene and even mandate prophylactic surgery.
INTRODUCTION In May 2013, the widely acclaimed Hollywood celebrity, Angelina Jolie, published an op-ed in The New York Times announcing that her mother, grandmother, and aunt had had cancer, that she had tested positive for the BRCA mutation, and that she had undergone bilateral prophylactic mastectomies to prevent breast cancer.1 This announcement led to what oncologists refer to as “The Angelina Jolie Effect,” a more than doubling in the demand for BRCA testing in women who would not otherwise have gone for testing, but were at high risk for carrying the mutation based on family history and therefore should have undergone genetic testing.2 In March 2015, Angelina Jolie published a second oped in The New York Times disclosing that she had undergone laparoscopic oophorectomy, removal of the ovaries to prevent ovarian cancer, and that she was receiving hormone replacement therapy to prevent the side-effects of premature menopause.3
SCIENTIFIC BACKGROUND Hereditary breast cancer accounts for 5%–10% of all breast cancer.4–7 The vast majority of inherited breast cancers are due to mutations in two breast cancer genes referred to as BRCA1 and 2.6 The risks of developing breast and ovarian cancer are higher in carriers of the BRCA1 mutation compared to carriers of BRCA2.8 In addition, cancer is more likely to occur at a younger age in carriers of BRCA1 mutations than in carriers of BRCA2. An average woman has a 12% lifetime risk of developing breast cancer.9 In a recent population-based study of Ashkenazi Jews in Israel, regardless of family history, the risks of developing breast cancer among BRCA1 and 2 carriers were 60% and 40%, respectively, and the risks of developing ovarian cancer were 53% and 62%, respectively.10 These results are consistent with the findings of a meta-analysis of 10 studies of patients in high-risk clinics which reported that the risks of developing breast cancer by the age of 70 in BRCA1 and 2 carriers are 57% and 49%, respectively, and the risks of ovarian cancer are 40% and 18%, respectively.8 These risks are significantly higher among women born more recently than among women born earlier, a birth cohort effect presumably due to modifications in non-genetic factors such as earlier menarche and later childbearing.10
Possible interventions for BRCA carriers might include increased surveillance, chemoprevention, and prophylactic surgery. Surveillance for breast cancer has consisted of MRI and mammogram beginning at age 25 or individualized to 10 years before the first cancer diagnosed in the family. While the addition of MRI to mammogram increases cancer detection rates and diagnoses cancer at an earlier stage, this strategy has not been shown to prolong survival in BRCA carriers.11–14 Surveillance for ovarian cancer has consisted of trans-vaginal ultrasound and blood tests for elevated tumor markers such as CA-125. However, this strategy has not been found to be effective.15,16 In fact, the National Cancer Institute does not recommend surveillance for ovarian cancer among BRCA carriers.
Another approach to reducing the risk of cancer among BRCA carriers is chemoprevention: tamoxifen to reduce the risk of breast cancer, and oral contraceptive pills to reduce the risks of ovarian cancer. Tamoxifen appears to be effective in reducing breast cancer in carriers of BRCA2 but not in carriers of BRCA1.17,18 The differential effect of tamoxifen may be due to the differential expression of the estrogen receptor in tumors of BRCA carriers. The BRCA1 carriers tend to develop estrogen receptor-negative breast cancers which do not depend on estrogen to grow, and the BRCA2 carriers tend to develop breast cancers that are estrogen receptor-positive and depend on estrogen to grow.19–21 Chemoprevention with tamoxifen has not been shown to prolong survival in BRCA2 carriers.17,18 Oral contraceptive pills have been shown to reduce the chances of developing ovarian cancer in BRCA carriers by 50% without increasing the risk of breast cancer, but this intervention has not been shown to reduce the chances of dying of ovarian cancer.22
PROPHYLACTIC SURGERY Prophylactic surgery consists of mastectomy, removal of the breasts to prevent breast cancer, and oophorectomy, removal of the ovaries to prevent ovarian cancer. Prophylactic mastectomy reduces the chances of developing breast cancer by 90% or more.23–30 In one series, there were no cases of breast cancer 3 years after prophylactic surgery.29 Prophylactic oophorectomy reduces the chances of developing ovarian cancer by 80% and can also reduce the chances of developing breast cancer by 50%.31–35 Carriers of the BRCA mutation may opt for mastectomy alone with surveillance of the ovaries (as Angelina Jolie chose to do between 2013 and 2015), prophylactic oophorectomy alone which will reduce the chances of developing both breast and ovarian cancer, or prophylactic mastectomy and oophorectomy (which Angelina Jolie ultimately chose to do). The ideal age to perform prophylactic oophorectomy is not known, but the recommendation is to complete childbearing by age 35–40 and then undergo prophylactic surgery as there is concern that delaying oophorectomy would increase the chances of developing ovarian cancer.35 In addition, the magnitude of the protective effects of oophorectomy in reducing the chances of breast cancer is greater the younger the age of oophorectomy.29,36 Oophorectomy at this age does cause premature menopause; however, as Angelina Jolie has illustrated, these symptoms can safely be managed with short-term hormone replacement therapy.37–39
How do BRCA carriers cope with prophylactic surgery? Emerging data would suggest that overall quality of life for BRCA carriers who undergo prophylactic surgery is not compromised by surgery. The BRCA carriers who undergo prophylactic surgery report high satisfaction with surgery and less cancer worry than BRCA carriers who opt for surveillance.40,41 However, prophylactic surgery may be associated with sexual dysfunction and menopausal symptoms which can be addressed by proper medical intervention. Although it was initially thought, based on theoretical modeling, that BRCA carriers who underwent prophylactic surgery would live longer than those who opted for surveillance, this was not based on actual prospective patient data.42 Recently, a number of studies have confirmed that BRCA carriers who undergo prophylactic surgery live longer than those who undergo surveillance.29,35,43 In one series, the chances of dying of ovarian cancer were reduced by 95% among BRCA carriers who opted for prophylactic oophorectomy compared to carriers who opted for surveillance, the chances of dying of breast cancer were reduced by 90%, and the chances of dying of any cause were reduced by 70%.29 Previously, it was assumed that prophylactic oophorectomy would reduce the chances of a BRCA carrier dying of ovarian cancer but that inducing premature menopause would be harmful and negate any beneficial effects of preventing ovarian cancer. Physicians believed that the BRCA carrier who opted for prophylactic oophorectomy was “trading” ovarian cancer for a host of new medical problems associated with entering premature menopause for no “net” benefit. Yet, the most recent studies show that, overall, BRCA carriers who undergo prophylactic surgery live longer than those who opt for surveillance, demonstrating that, regardless of what medical problems premature menopause may cause, the net effect of prophylactic surgery is that carriers who undergo such surgery live longer. The medical benefits clearly outweigh the risks.
HALACHIC ISSUES From a Halachic perspective, there are several concerns arguing against prophylactic surgery (Table 1). First of all, prophylactic surgery involves mutilation of a healthy organ. Secondly, removing a healthy organ—especially one that defines a woman’s sexuality and her appearance—may cause significant psychological distress, although the available quality of life data would suggest that from a psychological perspective this surgery is well tolerated.40,41
In addition, there are two other potential Halachic concerns regarding removal of ovaries. First, removal of ovaries may prevent the BRCA carrier from fulfilling the mitzvah to “be fruitful and multiply” (pru u’revu). Second, removal of ovaries may violate the prohibition against castration (sirus). Arguing in favor of prophylactic surgery are new, emerging, compelling medical data showing that BRCA carriers who undergo prophylactic surgery are less likely to die of cancer and more likely to live longer than women who opt for surveillance. Angelina Jolie’s decision prophylactically to remove her healthy breasts and most recently her healthy ovaries raises several Halachic questions including the following: (1) is there a Halachic obligation to prevent disease; (2) is it permitted for a BRCA carrier to remove a healthy organ; (3) does prophylactic surgery violate the prohibition against harming one’s body (chovel); (4) does prophylactic oophorectomy prevent the BRCA carrier from fulfilling the mitzvah to procreate; (5) does prophylactic oophorectomy violate the prohibition against castration; (6) is it permitted to perform prophylactic surgery in a BRCA carrier; (7) is a BRCA carrier obligated by Halacha to undergo prophylactic surgery; and, lastly, (8) are we as a Jewish society, particularly in Israel, obligated to pay for prophylactic surgery in BRCA carriers?
In summary, there is a clear Halachic obligation to prevent disease. It is permitted to remove a healthy body part to prevent disease in the future. Prophylactic oophorectomy interferes with obligations to procreate and prohibitions of castration. Oophorectomy after completing childbearing helps eliminate issues relating to “procreation.” When performed after menopause, prophylactic oophorectomy may obviate the prohibition against castration. Ultimately, saving a life overrides castration and any prohibitions including the prohibition against harming one’s body. Given the emerging data favoring prophylactic surgery, a growing number of Jewish arbiters believe that prophylactic surgery is Halachically permitted, with some positing that a BRCA carrier is obligated to undergo prophylactic surgery.
Angelina Jolie’s Status in Judaism Angelina Jolie’s very public medical journey has increased awareness of the BRCA mutation and the demand for testing in high-risk women who would not otherwise have been tested. In addition, she has increased interest in potentially life-saving prophylactic surgery. It is not possible to measure how many lives she has saved by making her very personal, medical odyssey public. Regardless of her other behaviors and politics, her decision to publicize her status as a BRCA carrier and her decisions to undergo prophylactic surgery make her worthy of the description in Sanhedrin, “Whoever saves one Jewish life is considered to have created an entire world.”63
Angelina Jolie has created many worlds, and for this we as a Jewish people must be eternally grateful.
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