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Department of Nutrition, University of California Davis, Davis, CA 95616
2To whom correspondence should be addressed. E-mail: bllonnerdal{at}ucdavis.edu.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • mammary gland • copper transport • lactation • zinc deficiency
Marginal zinc deficiency is more common than previously thought, and women and children are at particular risk for marginal Zn deficiency due to the combined effects of inadequate Zn intake and increased demand for Zn as a consequence of reproduction and growth (1,2). Copper plays an essential role as a cofactor for enzymes that generate cellular energy, cross-link connective tissue and mobilize cellular iron (3). Due to its ability to generate free radicals, excess Cu can be toxic; therefore, organisms have developed multiple mechanisms to tightly regulate Cu homeostasis and the availability of free Cu within the cell (4). Although few human studies have examined the effect of Zn intake on Cu metabolism, limited existing data suggest that a marginal Zn diet has profound negative effects on Cu balance and Cu status (5). Studies in rodent models have also documented that marginal Zn deficiency can result in reduced plasma Cu (6) and increased liver and kidney Cu (7), suggesting an effect of Zn deficiency on tissue Cu transport.
The fetus accumulates liver stores of Cu, which are rapidly mobilized after birth (8). Reinstein et al. (9) observed that offspring from rats fed a low Zn diet throughout pregnancy had higher total fetal and liver Cu than offspring from control dams, suggesting that they were exposed to elevated Cu levels in utero. The effect of low Zn intake on Cu metabolism may extend into the neonatal period because rats fed a marginal Zn diet throughout pregnancy and lactation had a trend toward higher milk Cu concentration compared with rats fed control (10). This suggests that infants from mothers consuming a low Zn diet during pregnancy and lactation may be exposed to elevated Cu concentrations both pre- and postnatally and they may be particularly at risk for the adverse effects of excess Cu as a consequence of the immaturity of neonatal Cu homeostasis (11). Although the mechanisms mediating the effects of Zn deficiency on Cu metabolism are currently unknown, these observations suggest that Zn deficiency may alter Cu transport in tissues such as the placenta and mammary gland.
Recently, several candidates for mammalian Cu transporters have been identified (12,13). Ctr1, a plasma membrane-associated Cu import protein (14), transports Cu with high affinity and is expressed in all tissues examined (12,13). Although the precise mechanism through which Ctr1 protein transports Cu is not yet known, it is believed to require multimerization of several Ctr1 proteins (14), and is endocytosed in response to Cu exposure (15). The Menkes Cu ATPase (ATP7A) belongs to the P-type ATPase family of transmembrane proteins, and mutations in the ATP7A gene result in impaired cellular Cu export (16). ATP7A expression is ubiquitous and its gene product is localized to a perinuclear region in transfected cells (17) and the mammary gland of mice and humans in the nonlactating state (18,19). However, mammary gland Atp7A expression is higher and relocalized during lactation, similar to its diffuse staining pattern in Chinese hamster ovary cells in response to Cu exposure (17), which suggests that mammary gland Atp7A plays a regulatory role in mammary gland Cu transport during lactation.
The Wilson Cu ATPase (ATP7B) also belongs to the P-type ATPase family and is homologous to ATP7A (20). Mutations in the ATP7B gene lead to impaired biliary excretion of Cu from the liver and subsequent hepatotoxicity (16). Atp7B is also localized to the trans-Golgi network and in hepatocytes, it translocates to a large vesicular compartment in response to increased Cu concentrations (20,21). A murine mutation in ATP7B (toxic milk, tx) results in defective mammary gland Atp7B translocation impairing Cu export into milk (
20% of normal), and leads to neonatal death from Cu deficiency (20), suggesting that it plays the major role in mammary gland Cu export into milk (20). Interestingly, unlike Atp7A, only the localization and not the amount of Atp7B protein in the mammary gland differs between lactating and nonlactating mice (20), suggesting different regulatory mechanisms in the mammary gland during lactation for these Cu transporters. In this study, we hypothesized that a marginal Zn diet during pregnancy and lactation, without the complications of severe Zn deficiency, specifically affects mammary gland Cu transporter expression, protein levels and localization and that these alterations result in high milk Cu levels, increased neonatal Cu intake and high tissue Cu levels.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Rats were fed a casein-based, semipurified, experimental diet based on the AIN-93 recommendation (22). The diet composition differed only in Zn content with the control diet containing 25 mg Zn/kg (CON) and the diet marginally low in Zn containing 10 mg Zn/kg (ZD), as previously described (23).
Animals.
This study was approved by Animal Research Services at the University of California, Davis, which is accredited by the American Association for the Accreditation of Laboratory Animal Care. Virgin Sprague-Dawley rats (n = 12; 250 g) were obtained commercially (Simonsen, Gilroy, CA) and maintained in stainless steel hanging cages until pregnancy was confirmed, at which time rats were moved to plastic cages containing shaved wood bedding. Six rats/diet were randomly assigned to consume control diet or the diet marginally low in Zn ad libitum. Rats were fed the diets for 70 d before mating, through gestation and until d 11 of lactation. Food intake was monitored throughout the study to confirm that Zn restriction did not result in food cycling. On postnatal d 2, litters were culled to 10 pups. On postnatal d 11, milk (3 mL/dam) was collected as previously described after oxytocin injection (subcutaneous, 10 IU/dam) (23). Whole blood was removed via cardiac puncture and collected into heparinized vials, and dams and pups were killed by asphyxiation with CO2. Plasma was separated by centrifugation at 2000 x g for 15 min at 4°C and frozen at -80°C until analyzed for Zn and Cu concentration and ceruloplasmin (Cp) activity. Mammary glands were dissected and immediately snap-frozen in liquid nitrogen for determination of Zn and Cu concentrations and Cu transporter protein levels, or homogenized in TriZOL (Life Technologies, Rockville, MD) for RNA isolation. Livers from dams and pups and small intestines from pups were dissected and immediately snap-frozen for determination of Zn and Cu concentrations. To eliminate immediate effects of hormonal alterations on Cu transporter localization, four additional control rats at d 11 of lactation were removed from their pups for 4 h; mammary glands were dissected and fixed in 4% paraformaldehyde in PBS, pH 7.4 for immunostaining.
Cu and Zn analysis.
Plasma, tissue and milk were digested as previously described (23), and Zn and Cu concentrations were analyzed by flame atomic absorption spectroscopy (Model Smith-Heifjie 4000, Thermo Jarrell Ash, Franklin, MA).
Ceruloplasmin activity.
Cp oxidase activity in plasma and milk was assayed with o-dianisidine dihydrochloride following the method of Schosinsky et al. (25).
Messenger RNA extraction and production of cDNA probes to CTR1, ATP7A and ATP7B by PCR.
Total mammary gland RNA was extracted with TriZOL (Life Technologies) following the manufacturer’s instructions. mRNA was isolated using the Microfast Track mRNA Isolation Kit (Invitrogen, Carlsbad, CA), and 1 µg mammary gland mRNA was used for cDNA synthesis by RT-PCR using oligo dT primers (cDNA cycle Kit, Clonetech, Palo Alto, CA). Primers used for cDNA synthesis were as follows: rat Ctr1 (Genebank Accession # NM 133600), 113GAA GAC CTT TGC GCT GAC TC132 and 504GAT GGT TCC ATT TGG TCC TG485 producing a 392-bp amplicon; rat ATP7A (Genebank Accession # NM 052803), 2240GGA TGT GCT GAT TGT GTT GG2259 and 2639ATT GCT TCC CCT GTG ATG AG2619 producing a 400-bp amplicon; rat ATP7B (Genebank Accession # NM 012511), 931TCA CCC CCT TGT TCC TAC AG950 and 1327GAA TTC CCA GAG CTG GAC TCG1308 producing a 400-bp amplicon. PCR cycling parameters were as follows: Ctr1: 95°C for 1 min, followed by 21 cycles at 94°C for 30 s and 68°C for 3 min; ATP7A: 95°C for 1 min, followed by 29 cycles at 94°C for 30 s and 68°C for 3 min; ATP7B: 95°C for 1 min, followed by 30 cycles at 94°C for 30 s and 64°C for 3 min with a final extension at 70°C for 3 min. PCR transcripts were separated by gel electrophoresis and purified as previously described (23). Glyceraldyde phosphatedehydrogenase cDNA (a generous gift from Katti Jessen, University of California, Davis) was used as a normalization control.
RNA analysis by Northern blotting.
cDNA probes were labeled with 32P (cDNA Labeling Kit, Amersham Pharmacia Biotech, Piscataway, NJ). Equal amounts of total RNA from individual mammary glands (20 µg) were denatured in MOPS sample buffer containing ethidium bromide (Sigma, St. Louis, MO) and electrophoresed, transferred and hybridized as previously described (23). Radiolabeled membranes were exposed for 2–8 d at -80°C in an autoradiography cassette with 2 enhancing screens (Fisher, Pittsburg, PA). Relative amounts of mRNA were quantified by densitometry using the Chemi-doc Gel Quantification System (Bio-Rad, Hercules, CA).
Production of antibodies to Ctr1, Atp7A and Atp7B.
Peptide fragments of rat Ctr1 (Genebank Accession # NP 598284; amino acids 90–110: LLRKSQVSIRYNSMPVPGPNG), rat Atp7A (Genebank Accession # AAB06393; amino acids 238–258: IKKQPKYLKLGAIDVERLKST) and rat Atp7B (Genebank Accession # NP 036643; amino acids 428–469: NITTNRVSSGNSVPQAVGDSPGSVQNMASDTRGLLTHQGPGYLSD) were synthesized (Genemed Synthesis, South San Francisco, CA) with an additional cysteine residue for conjugation to keyhole limpet hemocyanin (KLH) at the C-terminal end. Sequences were verified by amino acid analysis and MS. KLH-conjugated peptides were injected into New Zealand White rabbits (1 mg peptide/rabbit) for polyclonal antibody production. Specificity of peptide antibodies was verified by the appearance of specific bands not detected after incubation with preimmune serum on a Western blot of mammary gland protein (see below).
Preparation of mammary gland protein extracts and membrane fractions.
Mammary gland (250 mg) was homogenized in Hepes-EDTA buffer as previously described (23). The postnuclear supernatant was isolated by centrifugation for 5 min at 500 x g at 4°C. This supernatant was subjected to centrifugation at 15,000 x g for 10 min at 4°C (resulting in pellet 1), followed by 100,000 x g for 45 min at 4°C (resulting in pellet 2) then 100,000 x g for 4 h at 4°C (resulting in pellet 3) (26) to isolate crude fractions of large membrane vesicles, microsomes and small vesicles, respectively. The membrane pellets were resuspended in homogenization buffer and protein concentration was determined by the Bradford protein assay (Bio-Rad). Total mammary gland protein extracts, membrane fractions and the final supernatant were divided into aliquots and stored at -80°C until analysis by SDS-PAGE.
Immunoblotting.
Equal amounts of mammary gland protein (50–100 µg) were resolved by SDS-PAGE under reducing (Atp7A and Atp7B, 7.5%) or nonreducing conditions (Ctr1, 10%). Membrane protein was transferred to nitrocellulose for 90 min at 350 mA. Blots were blocked overnight at 4°C with 5 g/100 mL nonfat milk in PBS/Tween-20 (0.1 g/100 mL, PBS-T) and washed 3 times in PBS-T. Blots were incubated with primary antiserum for 1 h (Ctr1, 1:2000; Atp7A,1:6000;Atp7B,1:2500 in PBS-T), washed 3 times in PBS-T, then incubated with donkey-anti-rabbit IgG conjugated to horseradish peroxidase for 1 h (1:20,000 in 5 g/100 mL nonfat milk/PBS-T) and washed extensively with PBS-T. Antibody to ß-actin (Santa Cruz Biotechnology, Santa Cruz, CA) was used to control for protein loading. Blots were visualized with enhanced chemiluminescence (Super Femto Detection Reagent, Pierce Endogen, Rockford, IL) and quantified using the Chemi-doc Gel Quantification System (Bio-Rad).
Immunostaining of mammary gland.
Fixed mammary glands from control rats at d 11 of lactation were embedded and immunostained as previously described (23).
Statistical analysis.
Results are presented as means ± SD, n = 6 dams/diet or n = 60 pups/diet. Statistical comparisons were made using a t test (Prism Graph Pad, Berkeley, CA). Differences were considered significant at P < 0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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23, 28, 36, 65 and 90 kDa. We detected one major protein band that immunoreacted with Atp7A antiserum (180 kDa) and one major band at 170 kDa that immunoreacted with Atp7B antiserum, indicating that these antisera can differentiate Atp7A from Atp7B despite the large degree of homology between these proteins.
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5.4 kb; this was not affected by a marginal Zn diet. The levels of native Ctr1 proteins were altered to different extents in response to a marginal Zn diet (Fig. 4A and B). The level of the monomeric 23-, 28- and 36-kDa Ctr1 proteins in mammary gland extracts was higher in rats fed a marginal Zn diet than in rats fed the control diet (P < 0.05), whereas the dimeric 65-kDa and multimeric 90-kDa proteins were not affected. Using differential centrifugation of mammary gland extracts, we further characterized the intracellular distribution of Ctr1 (Fig. 5). No Ctr1 proteins were detected in the supernatant (very small vesicles and cytosol) separated from the final pellet (small vesicles). We determined that the 23-, 28-, 36- and 65-kDa proteins were present in all 3 pellets, whereas the 90-kDa protein was detected primarily in pellet 1 (large membranes). Although the 36- and 65-kDa proteins were distributed approximately equally among pellets, we detected more of the 23-kDa protein in pellet 2 (microsomes). This suggests that the majority of Ctr1 monomers and dimers is localized to a subcellular location that is different from the 90-kDa multimer. Furthermore, we determined that the distribution of the 36- and 65-kDa proteins was altered in response to a marginal Zn diet (Table 3). Approximately 75% of the 36- and 65-kDa proteins were detected in pellets 2 (microsomes) and 3 (small vesicles), and they were distributed approximately equally in these pellets in control rats. However, this distribution shifted in response to a marginal Zn diet, resulting in 20% in pellet 2 (microsomes) and 80% in pellet 3 (small vesicles), suggesting that vesicles containing Ctr1 are redistributed in response to marginal Zn intake.
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7 kb. However, rats fed a marginal Zn diet had higher ATP7A protein levels (Fig. 4C,D) than control rats. After differential centrifugation, 50% of the 180-kDa protein was detected in pellet 2, 30% in pellet 3 and 20% of mammary gland Atp7A was detected in pellet 1. Interestingly, the distribution of Atp7A in the mammary gland shifted significantly toward the fraction containing larger vesicles (pellet 2) in response to a marginal Zn diet (Fig. 6, Table 3).
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6 kb in the mammary gland during lactation. Rats fed the marginal Zn diet had higher ATP7B mRNA (Fig. 3A,B) and protein levels than rats fed control (Fig. 4C,D). After differential centrifugation, the majority of Atp7B protein in CON was found in pellet 3 with smaller amounts detected in the final supernatant and pellet 2 (Fig. 6, Table 3). However, there was no shift in Atp7B distribution detected in rats fed a marginal Zn diet. |
DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The precise mechanisms utilized by the mammary gland to import Cu from the maternal circulation, transport it through the mammary gland and export it into milk are currently unknown. Although ceruloplasmin has been suggested to transport Cu into tissues via a putative Cp receptor (3), aceruloplasminemic rats have apparently normal Cu metabolism (27) and there is no evidence to suggest that pups suckled from aceruloplasminemic dams suffer from Cu deficiency. Furthermore, recent data suggest that if Cp plays a direct role in mammary gland Cu import, it is a minor one (28). However, in accordance with other reports (5,29), plasma Cp activity was higher in rats fed a marginal Zn diet, implying that incorporation of Cu into Cp in the liver and secretion into systemic circulation is increased as a result of increased intestinal Cu absorption (5,29). An increase in intestinal Cu absorption transiently elevates systemic Cu levels before Cu uptake by the liver. Because Cu is rapidly and preferentially absorbed by the mammary gland before Cu uptake and incorporation into Cp by the liver (28), this high Cp activity indicates that the mammary gland of rats fed a marginal Zn diet may be exposed to transiently higher circulating Cu levels and that Cp activity might be a useful marker of marginal Zn status.
To our knowledge, this is the first report to document the expression of Ctr1 in the mammary gland. We determined that the mammary gland in the lactating rat expresses 1 CTR1 transcript unlike what has been observed for rat liver and intestine (13). Similar to what was observed for CTR1 expression in response to Cu deficiency in rat liver, intestine and hypothalamus (13), the expression of mammary gland CTR1 was not affected by a Zn-deficient diet. Studies in transfected cells suggest that human Ctr1 monomers and dimers of 30 and 60 kDa form a 90-kDa homomultimeric complex, and it has been postulated that multimerization may be necessary to transport Cu (14,30). In accordance with these observations, we also detected multiple native Ctr1 proteins of various sizes by Western blot under nonreducing conditions. We hypothesize that these multiple protein sizes are different post-translationally processed forms of Ctr1, including the 23-kDa core protein (30,31), several glycosylated products (28 and 36 kDa), dimers (65 kDa) and multimers (90 kDa) as has been observed for yeast and human Ctr1 (30,31). Ctr1 in the mammary gland is localized to the plasma membrane and within vesicular compartments as has been observed for various cell lines (31); results from this study suggest that the monomeric and dimeric proteins are found primarily in smaller vesicles and that multimerization of the 90-kDa complex occurs in a fraction containing larger vesicles. Although this suggests that multimerization occurs at the plasma membrane, this hypothesis remains to be addressed.
Despite a lack of significant effect on mammary gland CTR1 mRNA level, rats fed a marginal Zn diet had higher protein levels of the monomeric 23-kDa Ctr1 backbone, 28- and 36-kDa glycosylated Ctr1 proteins, although no reproducible increase in the larger dimeric or multimeric complex was observed. Interestingly, differential centrifugation revealed a potential shift in the localization of mammary gland Ctr1 in response to a marginal Zn diet, with the rats fed a marginal Zn diet having a greater percentage of the 36- and 65-kDa proteins in the fraction comprised of smaller vesicles. These results suggest that mature Ctr1 in the mammary gland may undergo endocytosis in response to a marginal Zn diet as has been shown to occur in transfected Hek293 cells in response to physiologic levels of Cu exposure (15). However, the possibility that this shift in Ctr1 localization is a result of altered vesicle traffic in response to Zn deficiency cannot be eliminated, and our current results do not suggest that mammary gland Ctr1 is degraded as a consequence of internalization in response to marginal Zn intake as has been observed in response to Cu exposure (15). Studies are currently underway to determine whether these changes in Ctr1 proteins and localization in the mammary gland are specific effects of alterations in intracellular Zn or Cu pools or extracellular signals. There is precedence for specific effects of Zn on Cu transport because Caco-2 cells respond to elevated Zn concentrations by increasing Cu uptake and decreasing export, suggesting that Zn may play a role in the regulation of Cu transporters (32) at some currently unknown level.
In accordance with what others have reported (19,33), we detected one ATP7A transcript in the mammary gland; its level was not affected by Zn intake. We determined that in the mammary gland during mid-lactation, Atp7A is localized primarily in association with the plasma membrane. In its role as a Cu exporter (34), our documentation of its localization to the luminal membrane of mammary epithelial cells would predict that an increased amount of Atp7A protein may directly result in an increased amount of Cu exported into milk (15,17). In addition to higher protein levels, our data suggest that the altered Atp7A localization in rats fed a low Zn diet, away from smaller vesicles (26), may also have contributed to the higher milk Cu concentration observed in these rats. The mechanisms through which low Zn intake affects mammary gland Atp7A protein levels and localization are currently unknown. However, because the relocalization of Atp7A we observed is reminiscent of the effects of high extracellular Cu exposure on Atp7A localization (35), the mammary gland may be responding to increased exposure to circulating Cu. Alternatively, because mammary gland Atp7A levels are higher during lactation than in a nonlactating state (19), mammary gland Atp7A levels may be responding to the physiologic effects of marginal Zn intake on lactation such as alterations in hormonal signaling. Studies are currently underway to examine this possibility.
A third Cu transporter that has been identified in the mammary gland is ATP7B (20). A mutation in ATP7B that prohibits its translocation from the trans-Golgi network to an intracellular vesicular compartment is believed to be responsible for the phenotypically low milk Cu concentration observed in tx mice (20) and suggests that Atp7B is the major contributor to milk Cu concentration. Both ATP7B mRNA and protein levels were higher in rats fed the marginal Zn diet than in rats fed the control diet; however, there was no change in Atp7B localization as was observed in cells exposed to excess Cu (36). Because Atp7B participates in Cu incorporation into Cp in the liver (37), we hypothesize that the high mammary gland Atp7B level was most likely responsible for the high milk Cu and Cp activity levels. The fact that plasma Cp activity was also higher in these rats suggests that this response is not specific to the mammary gland, and further studies are currently underway to examine this hypothesis. Although these changes may be a consequence of subtle intracellular changes in Zn or Cu pools because mammary gland metallothionein levels were also reduced in these rats (38), the mechanisms through which a low Zn diet affects ATP7B mRNA and protein levels remain to be elucidated. There is precedence for an effect of Zn on Atp7B in that high Zn concentrations inhibit tertiary conformation and ATPase activity in vitro (39). However, effects of high or low Zn concentrations on ATP7B in vivo have yet to be demonstrated.
In addition to our observations that a maternal diet marginal in Zn directly affects mammary gland Cu metabolism, downstream effects of negative functional outcomes in pups from dams fed the marginal Zn diet during pregnancy and lactation were also observed. Although secondary effects of fetal Zn restriction cannot be eliminated, we hypothesized that the alterations we observed in neonatal Cu metabolism result primarily from increased Cu intake from elevated milk Cu levels. In accordance with observations made by others (6,7,9), pups suckled from dams fed a marginal Zn diet had lower plasma Zn and Cu concentrations than pups from control dams, suggesting that low maternal Zn intake, even without overt indications of Zn deficiency, induces a secondary Zn and Cu "deficiency" in the neonate. However, a caveat to this supposition of "deficiency" is that we observed threefold higher Cu concentration in the small intestine of these neonatal rat pups, suggesting that the intestine is sequestering Cu, which may functionally protect the neonate from increased Cu exposure. Interestingly, although plasma Cu was lower in pups from dams fed the marginal Zn diet, their plasma Cp activity was not affected. Although there is little known about the regulation of neonatal Cu metabolism, studies are currently underway to determine whether these effects on Cp activity are a consequence of "immature" Cu regulation. Our observations that small intestine and liver Zn concentrations were higher in pups from dams fed the marginal Zn diet in combination with a lower plasma Zn level suggest that Zn transport mechanisms may be compromised, facilitating the sequestration of Zn in both of these tissues. Studies to determine whether this is a primary result of neonatal Zn deficiency or a secondary effect of increased Cu exposure are currently underway.
In summary, a marginal Zn diet during pregnancy and lactation had pronounced effects on Cu transporters in the mammary gland without significant effects on maternal Zn or Cu status. The results from this study suggest that the higher level of mammary gland Cu transporters in addition to the relocalization of Atp7A led to a higher milk Cu concentration compared with control rats, and further relocalized Ctr1 into smaller vesicles, possibly as a result of endocytosis. The fact that plasma and milk Cp activity were both higher in rats fed a marginal Zn diet suggests that this effect may not be mammary gland specific. Furthermore, marginal maternal Zn intake resulted in increased neonatal Cu intake as a consequence of increased milk Cu concentration; this ultimately resulted in neonatal Cu and Zn accumulation in the small intestine and liver, leaving the neonate at risk for consequences of excess Cu intake. Whether this occurs in humans is not yet known.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: CON, control; Cp, ceruloplasmin; KLH, keyhole limpet hemocyanin; PBS-T, PBS-Tween-20; ZD, zinc deficient.
Manuscript received 20 March 2003. Initial review completed 1 April 2003. Revision accepted 22 April 2003.
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