Localization and expression of BCAT during pregnancy and lactation in the rat mammary gland

Armando R. Tovar1, Enrique Becerril1, Rogelio Hernández-Pando2, Gabriel López1, Agus Suryawan3, Soledad Desantiago1, Susan M. Hutson3, and Nimbe Torres1

1 Departamento de Fisiología de la Nutrición and 2 Departamento de Patología, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Mexico City, 14000 Mexico; and 3 Department of Biochemistry, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157


    ABSTRACT
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INTRODUCTION
MATERIALS AND METHODS
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During lactation, branched-chain aminotransferase (BCAT) gene expression increases in the mammary gland. To determine the cell type and whether this induction is present only during lactation, female rats were randomly assigned to one of three experimental groups: pregnancy, lactation, or postweaning. Mammary gland BCAT activity during the first days of pregnancy was similar to that of virgin rats, increasing significantly from day 16 to the last day of pregnancy. Maximal BCAT activity occurred on day 12 of lactation. During postweaning, BCAT activity decreased rapidly to values close to those observed in virgin rats. Analyses by Western and Northern blot revealed that changes in enzyme activity were accompanied by parallel changes in the amount of enzyme and its mRNA. Immunohistochemical studies of the mammary gland showed a progressive increase in mitochondrial BCAT (mBCAT)-specific staining of the epithelial acinar cells during lactation, reaching high levels by day 12. Immunoreactivity decreased rapidly after weaning. There was a significant correlation between total BCAT activity and milk production. These results indicate that the pattern of mBCAT gene expression follows lactogenesis stages I and II and is restricted to the milk-producing epithelial acinar cells. Furthermore, BCAT activity is associated with milk production in the mammary gland during lactation.

branched-chain amino acids; lactogenesis


    INTRODUCTION
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INTRODUCTION
MATERIALS AND METHODS
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PREGNANCY AND LACTATION require metabolic adaptations to meet the energy and amino acid requirements for both the mother and the infant. Amino acids from dietary protein are required for the synthesis of maternal and fetal tissues during pregnancy and after birth to support milk production during lactation. During lactation, the increased blood flow to the mammary gland and the high rate of amino acid extraction greatly increase the general pool of amino acids in the gland (38, 45). Extraction of the indispensable branched-chain amino acids (BCAA) from the blood actually exceeds the amount of these amino acids required for milk and tissue protein synthesis (43). Changes in the metabolism of leucine have been documented in pregnant women (14), and it has been shown that there are changes in the partitioning of BCAA oxidative capacity among different organs and tissues in the lactating rat (7).

It has been hypothesized that the lactating mammary gland is an important site of BCAA metabolism in the rat (44). BCAA, leucine, isoleucine, and valine are transaminated by branched-chain aminotransferase (BCAT; EC 2.6.1.42) to produce their respective branched-chain alpha -keto acids (BCKA). These keto acids are oxidatively decarboxylated by the BCKA dehydrogenase enzyme complex (BCKD). Two BCAT isoenzymes are found in mammals, a mitochondrial (mBCAT) and a cytosolic BCAT (10); only mBCAT is expressed in mammary gland (7). We have shown that, at peak lactation, there are significant increases in mBCAT mRNA, protein, and activity that are consistent with induction of mBCAT gene expression in the lactating rat (7). Indeed, the capacity of the mammary gland to transaminate BCAA exceeded that of skeletal muscle, and changes in activity were also associated with increased transamination of BCAA in mammary tissue (7, 8). We hypothesized that changes in mBCAT expression would reflect changes in gene transcription induced by hormonal changes occurring in the gland during pregnancy and lactation rather than increased proliferation of a specific cell population. In addition, we predicted that mBCAT would increase in response to an increase in demand brought about by increased milk production. To test these hypotheses, we investigated the time course of the changes in mBCAT mRNA, protein, and activity and determined the cellular localization of mBCAT during pregnancy, lactation, and weaning in the rat.


    MATERIALS AND METHODS
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Animals and experimental design. Sixty-five female Wistar rats with a mean weight of 239 ± 8 g were obtained from the animal research facility at the Instituto Nacional de Nutrición. Animals were housed in individual stainless steel cages in air-conditioned rooms. Lights were on between 0700 and 1900. Rats had free access to an 18% casein powdered diet (Teklad Test Diets, Madison, WI) and water throughout the study. Five virgin rats were killed and served as controls. The remaining 60 rats were mated, and gestational age was determined by vaginal smear to detect spermatozoa. These rats were divided randomly into three groups: 1) pregnant, 2) lactating, and 3) postweaning. Thirty-five rats were assigned to the pregnant group, and five rats each were killed on days 1, 5, 10, 14, 16, 18, and 20 of pregnancy. Twenty rats were assigned to the lactating group. After normal pregnancy and delivery, litter size was adjusted to 8 pups/dam. Litters were weighed during the lactation period to calculate milk production (g/day) according to Sampson and Jansen (31).

The day of birth was considered day 0 of lactation, and five rats each were killed on days 1, 5, 10, and 12 of lactation. Ten rats were assigned to the postweaning group, and five rats each were killed on days 1 and 2 after weaning. Litter size was also adjusted to 8 pups/dam during lactation. The protocol of this study was approved by the Ethics Committee in Animal Experimentation of the Instituto Nacional de Ciencias Médicas y Nutrición.

Tissue preparation. Rats were anesthetized with 30 mg/kg pentobarbital sodium before decapitation. Animals were killed between 0800 and 0930. All tissue used for RNA preparation was removed rapidly, frozen immediately with clamps precooled in liquid nitrogen, and stored at -80°C until used for the determination of mBCAT mRNA. The rest of the tissue was divided and used for measurement of BCAT activity and DNA content and for immunoblotting and immunohistochemistry. Tissue used for BCAT activity measurements and immunoblotting was blotted and weighed. The tissue was suspended in extraction buffer (1 g tissue/4 ml extraction buffer) containing 0.225 M mannitol, 0.075 M sucrose, 0.10 mM EDTA, and 5 mM MOPS (pH 7.0), 5 mM benzamidine, 1 mM diisopropyl fluorophosphate, 1 mM EDTA, 1 mM EGTA, 1 mM leupeptin, 5 mM dithiothreitol, and 10 ml/l Triton X-100. The tissue was minced and then homogenized with a Polytron (Kinematica, Littau/Lucerne, Switzerland) at the minimum setting. Tissue homogenates were centrifuged at 30,000 g for 1 h at 4°C to obtain tissue extracts. The supernatants could be stored at -80°C for 2 wk without loss of BCAT activity. Protein was determined by the biuret reaction in the presence of 2.5 mg/l sodium deoxycholate, with crystalline bovine albumin as standard.

Determination of BCAT activity. BCAT activity was assayed in all extracts by the method described by Hutson et al. (12) in small test tubes (10 × 45 mm) with a side arm (49). Activity was measured at 37°C in 50 mM potassium phosphate buffer, pH 7.8, containing 50 µM pyridoxal phosphate and 4 g/l 3-[(3-cholamidopropyl)dimethylamioniol]-1-propanesulfonate in a final volume of 1.0 ml. Fifty microliters of the supernatant were used, and the reaction was initiated by adding 100 µl of a 1.0 mM alpha -keto-[1-14C]isocaproate/12 mM isoleucine stock solution. alpha -Keto-[1-14C]isocaproate was prepared from L-[1-14C]leucine (Du Pont-NEN, Boston, MA) according to the procedure of Rüdiger et al. (30). After 5 min, the reaction was stopped by addition of 500 µl of 2 M sodium acetate, pH 3.4. The remaining alpha -keto-[1-14C]isocaproate was chemically decarboxylated by addition of 250 µl of 30% hydrogen peroxide. A 250-µl sample of the reaction mixture was added to a scintillation vial. [14C]leucine formation was determined by liquid scintillation counting of an aliquot of the reaction mixtures in biodegradable counting scintillant cocktail (Amersham, Buckinghamshire, UK). Each assay was performed in duplicate. A unit of enzyme activity was defined as 1 µmol of leucine formed/min at 37°C.

DNA content. Nucleic acids were extracted from samples of each killing time with perchloric acid (32), and DNA content was determined according to Giles and Myers (9). Cell number was calculated by dividing the DNA content per gram of tissue using the following formula (48)
no. of cells (millions) = <FR><NU>mg DNA × total organ × 1,000</NU><DE>6.2</DE></FR>

SDS-PAGE and immunoblotting. Proteins in the tissue extracts (40 mg; n = 3 rats) were separated by SDS-PAGE in 10% gels according to Laemmli (17). Before electrophoresis, all samples were boiled for 2 min in the presence of 1% SDS with 5% beta -mercaptoethanol. Premixed low-range protein molecular weight markers were obtained from Boehringer Mannheim (Indianapolis, IN). For immunoblotting, proteins in the SDS-PAGE gel were transferred to polyvinylidene difluoride (PVDF) membranes (Boehringer Mannheim, Mannheim, Germany) by use of a Transphor electrophoresis unit (Hoefer Scientific Instruments, San Francisco, CA). The PVDF membranes were blocked with 1.5% gelatin-1.5% albumin for 2 h at 37°C, and immunoblotting was performed using an anti-rat mBCAT-specific IgG fraction (10). The mBCAT-specific IgG fraction (1:2,500) was incubated for 1.5 h at room temperature, and immunoreactive protein bands were visualized using horseradish peroxidase-labeled goat anti-rabbit antibody (1:6,000) after the oxidation of luminol as luminescent substrate. Light emission was detected by exposure to autoradiography film (ECL, Amersham Life Science).

Preparation of total RNA and Northern blot analysis. Total RNA was isolated from mammary tissue as in Ref. 2. For Northern analysis, 20 µg of RNA were subjected to electrophoresis in a 1.5% agarose gel containing 37% formaldehyde and were transferred to Hybond-N+ (Amersham) and cross-linked with an ultraviolet cross-linker (Amersham). mBCAT mRNA was detected using a partial rat mBCAT cDNA probe (1, 13). The probe was a 900-bp PstI-EcoRI fragment of the rat mBCAT cDNA labeled with deoxycytidine 5'-[alpha -32P]triphosphate (3,000 Ci/mmol, Amersham) by use of the Rediprime DNA labeling system (Amersham). Filters were prehybridized with rapid-hyb buffer (Amersham) at 65°C for 45 min and then hybridized with the labeled probe for 2.5 h at 65°C. Membranes were washed once with 2× standard sodium citrate (SSC; 1× SSC = 0.15 M sodium chloride and 15 M sodium citrate)/0.1% SDS at room temperature for 20 min and then washed twice with 0.1× SSC/0.1% SDS at 65°C for 15 min each. The images were digitized, and radioactive bands were quantified using the Instant Imager electronic autoradiography system (Packard Instrument, Meriden, CT). Membranes were also exposed to Extascan film (Kodak, Guadalajara, Mexico) at -70°C with an intensifying screen. Results are expressed as arbitrary units.

Histology and immunohistochemistry. For light microscopy, tissue samples were fixed by immersion in absolute ethanol for 24 h, paraffin embedded, sectioned at 6 µm, and stained with hematoxylin and eosin. Immunohistochemical detection of the mitochondrial isoform of BCAT was performed with the rabbit anti-rat mBCAT IgG fraction or an immunoaffinity-purified anti-human mBCAT antibody. The specificity of the rat mBCAT polyclonal antibody has been established previously (13). For preparation of the immunoaffinity-purified human mBCAT antibody, the purified recombinant human protein (3) was attached to cyanogen bromide-activated Sepharose according to manufacturer's instructions (Pharmacia, Uppsala, Sweden) and was used for immunopurification of the antibodies as described in Wallin et al. (46). Before incubation with the primary antibody, endogenous peroxidase activity was inactivated with 0.03% H2O2 in absolute methanol, and tissue sections were then mounted on Silane-coated slides. The tissue mounted on the slide was incubated with PBS containing 3% BSA for 10 min. The slide was then incubated with anti-rat mBCAT IgG fraction (1:500) or anti-human mBCAT antibodies (1:250) in PBS containing 3% BSA overnight at 4°C. The next day, the tissue on the slide was washed three times with PBS containing 3% BSA for 1 min before incubation with goat anti-rabbit antibody conjugated with peroxidase (1:500) for 1 h. The slide was rinsed three times with PBS. Bound antibodies were detected with 3,3-diaminobenzidine, and the tissue was counterstained with hematoxylin. Negative controls consisted of replacing the primary antibody with normal goat sera. Immunoadsorbed controls were also performed with the human antibody. The antibody was incubated with a 50-fold excess of purified human mBCAT overnight at 4°C.

Statistical analysis. Data were analyzed by one-way ANOVA, and significant differences were identified by means of Fisher's protected least significant difference test (StatView version 4.02, Abacus Concepts, Berkeley, CA). Differences were considered significant at P < 0.05.


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BCAT activity, protein, and mRNA levels. To investigate the developmental expression of mBCAT in the mammary gland, BCAT activity, protein, and mRNA levels were measured on days 1, 5, 10, 14, 16, 18, and 20 of pregnancy, on days 1, 5, 10, and 12 of lactation, and on days 1 and 2 after removal of the pups at peak lactation (day 12). The results are summarized in Figs. 1-3. BCAT activity expressed as units per gram wet weight of tissue is shown in Fig. 1A; total BCAT activity (U/tissue) in the gland, with account taken for the changes in gland mass, is shown in Fig. 1B. BCAT activity (U/g wet tissue) and total BCAT activity increased significantly during the latter half of pregnancy, beginning on day 16, and a further increase in BCAT activity occurred in the gland after parturition. By day 5 of lactation, BCAT activity (U/g tissue) was eightfold higher than in the virgin gland. At peak lactation, BCAT activity (U/g tissue) was 15-fold higher than in the virgin mammary gland. Values for total BCAT activity were 70-fold higher than in the virgin control on day 12 of lactation (Fig. 1B). Removal of the pups on day 12 resulted in a rapid decline in BCAT activity. At 24 h after weaning, BCAT activity decreased by ~60% from the values at peak lactation. By 48 h, activity (U/g tissue) was nearly at the levels found in the virgin rat, whereas total BCAT activity was lower than at the end of pregnancy (41%) but higher than in the virgin gland. Thus the rapid decline in BCAT activity coincided with milk stasis in the gland. Protein content of the mammary gland increased during pregnancy, and at peak lactation on day 12, it was 4.8-fold higher than in control virgin rats (7). BCAT specific activity (mU/mg protein) in the gland was also measured, and the results are shown in Fig. 1C. The pattern of changes in BCAT specific activity was similar to the results expressed as units per gram wet tissue and per gland (Fig. 1, A and B); however, the magnitude of the changes was somewhat lower. During the first 10 days of pregnancy, BCAT specific activity did not differ significantly from that of virgin rats (Fig. 1C). After day 10, the specific activity of BCAT in the gland increased, reaching a plateau between day 16 and day 20 of pregnancy. During this period, corresponding to lactogenesis stage I, BCAT specific activity was ~114% higher than activity measured on day 1 of gestation and was 96% higher than on gestation day 10 (P < 0.01). BCAT specific activity increased rapidly after parturition, with the highest activity at peak lactation, 3.2-fold higher than in virgin rats, and dropped precipitously at weaning. After 2 days postweaning, BCAT specific activity was only 80% higher than in virgin controls but was 28.5% lower than at parturition (day 20). BCAT activity expressed as units per cell did not change during the first 10 days of pregnancy (Fig. 1D). By day 5 of lactation, BCAT activity (in U/cell) was 83% higher than in the virgin gland, reaching a value 1.1-fold higher than in the virgin gland at peak lactation.


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Fig. 1.   Time course of branched-chain aminotransferase (BCAT) activity in virgin, pregnant, lactating, and postweaning mammary gland expressed as: U/g wet tissue (A), U/total tissue (B), mU/mg protein (C), U/cell × 103 (D). Results are expressed as means ± SE.



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Fig. 2.   Immunoblotting with mitochondrial BCAT (mBCAT) antiserum. Top: 40 µg of the supernatant protein from kidney and from rat mammary gland during pregnancy, lactation, and postweaning were subjected to 10% SDS-PAGE, transferred to a polyvinylidene difluoride membrane, and immunoblotted using rat anti-mBCAT-specific IgG. Bottom: mBCAT protein concentration estimated by densitometry. Different letters indicate significant differences (P < 0.05).



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Fig. 3.   Northern blot analysis of mBCAT. Top: 20 µg of total RNA from kidney and from rat mammary gland during pregnancy, lactation, and postweaning were subjected to electrophoresis, blotting, and hybridization with 32P-labeled mBCAT cDNA probe. Bottom: concentration of mBCAT mRNA estimated by use of Instant Imager electronic autoradiography system. Different letters indicate significant differences (P < 0.05).

The amount of mBCAT protein was estimated by Western blotting, and equal amounts of tissue protein were loaded onto the gel (Fig. 2). Changes in mBCAT protein during pregnancy, lactation, and weaning followed a pattern similar to that of BCAT specific activity. During the first 10 days of gestation, the enzyme protein concentration did not change significantly. After day 16 of pregnancy, the concentration approximately doubled, similarly to the enzyme activity on day 20 compared with day 10 of pregnancy and virgin controls. During lactation, mBCAT protein concentration increased dramatically between days 1 and 5 of lactation. By day 12 (peak lactation), mBCAT protein levels were ninefold higher than in virgin rats. There was a rapid decline in the levels of mBCAT protein within 24 h after removal of the pups, although by 48 h the amount of protein was significantly higher than levels found in control virgin and pregnant rats.

Northern blot analysis was used to estimate mBCAT mRNA levels in mammary tissue (Fig. 3). The results are consistent with the data obtained for mBCAT protein and activity. During the first 10 days of lactation, mRNA abundance did not change significantly. After day 10, mBCAT mRNA concentration increased, and by day 20 of pregnancy, mBCAT mRNA concentration increased by 70% compared with day 10 of gestation. During lactation, mBCAT mRNA concentration increased steadily, reaching its highest expression levels at peak lactation that was 4.5- and 1.9-fold higher than in virgin and day 20 pregnant rats, respectively. After 24 h postweaning, mBCAT mRNA concentrations decreased to levels significantly lower than in virgin control rats. Thus the fall in mBCAT mRNA after weaning appeared to be greater than the fall in mBCAT protein and activity.

Immunohistochemical localization of mBCAT in the mammary gland. To determine the cellular location of mBCAT in the mammary gland, immunohistochemical analysis was performed on sections of the mammary gland isolated from virgin rats, from rats at days and 10 of pregnancy, at days 1, 5, 10, 14, 16, 18, and 20 of lactation, and at 24 and 48 h after weaning. In the virgin rat mammary gland, mBCAT immunostaining was at or near background in the ducts, whereas no staining was found in the connective tissue that surrounds the epithelial elements (Fig. 4A). During pregnancy, extensive proliferation of epithelial ducts was observed. These structures are located in the connective tissue that surrounds the numerous lobules constituted by growing alveoli with small lumina (Fig. 4, B and C). At days 5 and 10 of pregnancy, the ducts exhibited little mBCAT-specific staining. On day 20 of pregnancy, mBCAT-specific staining could be seen in the lobules of the acinar epithelium (Fig. 4D). During lactation, the mammary gland showed larger lobules constituted by numerous alveoli with dilated lumina. Lactating alveoli are formed by cylindrical epithelial cells, with cytoplasmic vacuoles representing spaces where cytoplasmic lipid droplets have been extracted during the embedding process. These cells showed a progressive increment in mBCAT-specific immunostaining during lactation. Epithelial cells that line the ducts were negative to mBCAT (Fig. 4E). The most intense staining was observed at day 12 of lactation (Fig. 4E). As shown in Fig. 4F, immunohistochemical staining of the gland at day 12 with mBCAT antibody preabsorbed with purified human mBCAT antigen showed only background staining. At 24 and 48 h after weaning, an accentuated and progressive decrement of immunoreactivity was seen (Fig. 4, G and H). By 48 h, only occasional cells exhibited mBCAT-positive immunostaining. These results indicate that increased expression of mBCAT in the lactating rat is localized in the milk-producing cells of the acinus.


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Fig. 4.   Immunolocalization of mBCAT in virgin, lactating, and postweaning mammary gland. a: in the paraffin sections, staining was below the limit of detection in epithelial duct cells in mammary gland of virgin rats (×100). b: on day 5 of pregnancy, numerous lobules constituted by acinar cells without immunoreactivity against mBCAT were observed (×100). c: on day 20 of pregnancy, some acinar cells with strong immunoreactivity to mBCAT were observed (×200). d: on day 1 of lactation, more acinar cells were immunoreactive (×200). e: at peak of lactation (day 12), lactating mammary gland showed intense immunostaining against mBCAT in lobular acinar cells (arrows). No immunostaining was observed in ductal epithelial cells (arrowheads) (×100). f: there was no immunostaining in mammary gland tissue on day 12 of lactation when mBCAT antibodies were preadsorbed with purified mBCAT (×100). g: mammary gland from day 1 postweaning rats showed a pronounced decrease in the acinar cell immunostaining (×200). H: after 2 days postweaning, most acinar cells were negative (×200).

Association between milk production and BCAT activity. As shown in Fig. 5, milk production was significantly correlated (r = 0.98; P > 0.001) with the total BCAT activity of the mammary gland (U/tissue). The range of milk production increased from 10 g/day on day 1 of lactation to 30-35 g/day at the peak of lactation.


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Fig. 5.   Association between milk production (g/day) and total BCAT activity (U/tissue) in the lactating mammary gland of the rat.


    DISCUSSION
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ABSTRACT
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The results presented in this study are consistent with the hypothesis that changes in mBCAT gene expression are associated with both changes in the number of alveolar cells of the gland and increased milk synthetic activity. The results in Figs. 1-4 show that mBCAT expression begins during the last phase of pregnancy, corresponding to lactogenesis stage I (beginning on day 14 of pregnancy), reaching high levels of expression at peak lactation on day 12. Interestingly, there was little change in activity between day 20 of pregnancy and day 1 of lactation, which corresponds to lactogenesis stage II. In agreement with our earlier results (5), at peak lactation, the level of mBCAT-specific mRNA was three- to fourfold higher, and there was a ninefold increase in the amount of mBCAT protein over levels found in the virgin gland. Because both the RNA and the protein content of the mammary gland increase dramatically during lactation (4), the actual total increases in mBCAT-specific mRNA and in mBCAT protein per gram of wet weight of tissue are in the order of ~100- and 50-fold, respectively (7). After weaning, mammary gland mBCAT mRNA returned within 24 h to levels found in the virgin rat. Changes in mBCAT mRNA were followed by a slower decline in mBCAT activity and protein. Thus the rapid decline in mBCAT mRNA and protein at weaning are likely to be associated with milk stasis.

The mammary gland consists of different cell types that change markedly during pregnancy and lactation (28). The number of nuclei per alveolar section remains relatively constant during pregnancy, but there is an approximately threefold increase in total glandular tissue (21) in the rat mammary gland during the second half of pregnancy. The increase in BCAT activity in late pregnancy probably reflects, in part, an increase in glandular tissue. Between days 10 and 20 of pregnancy, a modest doubling in mBCAT protein content and an approximately threefold increase in total BCAT activity were measured (Fig. 1). The immunohistochemistry studies showed that the increase in BCAT activity was exclusively associated with a rise in mBCAT-specific staining in the alveolar epithelial cells at the end of pregnancy. Background levels of mBCAT-specific staining could be seen in the virgin gland and up to day 10 of pregnancy. Thus the first small increases in BCAT activity, amount of enzyme, and mRNA concentration occur after day 14 of pregnancy and correspond to lactogenesis stage I. The second and more dramatic increase in mBCAT-specific immunostaining occurred after parturition. At parturition, progesterone withdrawal induces rapid development of milk secretion. There is an increase in alveolar diameter and a fall in alveoli per unit area in the gland during lactation (21). In the rat, total alveolar cell numbers also increase steadily during lactation. Thus, when total tissue BCAT activity was expressed per cell and the DNA content during pregnancy and lactation was considered, there was an approximate doubling, indicating that the increase in activity was related mostly to the increase in alveolar cells in the gland. Onset of lactation resulted in an intense mBCAT-specific staining in the secretory alveolar cells. mBCAT was localized in the secretory epithelium, and the changes in mBCAT content in these cells with the onset of lactation are consistent with induction of the enzyme. In the alveolar cells, mBCAT staining was found throughout the cytoplasm. This staining pattern is consistent with the distribution of mitochondria in lactating mammary epithelial cells (23). Histochemical results showed a rapid disappearance of the enzyme in the mammary tissue after weaning, associated with a fast decrease in mBCAT mRNA, amount of protein, and BCAT activity.

The increased expression of mBCAT during lactation is probably related to the accelerated anabolism occurring in the mammary gland for the synthesis of the different milk components. Gene expression of lipogenic enzymes in the mammary gland shows a time course pattern similar to that of mBCAT in different species during lacation (20). In fact, the biosynthetic capacity of fatty acids in the gland shows a pattern parallel to that of the lipogenic enzymes (20). Lactose synthesis in the mammary gland of the rat shows a similar trend and is also dependent on an increase in galactosyltransferase activity (47). Thus the increase in activity of these enzymes is related to the metabolic functions of secretory epithelial cells in the lactating gland to support the synthesis of the different milk components. mBCAT seems to be associated with this purpose, because total BCAT activity was significantly associated with milk production (Fig. 5).

The role of mBCAT in the mammary gland during lactation is not clear, however. BCAA are actively transported into the mammary gland through the L system (34). Large neutral amino acids (LNAA), including BCAA, are not accumulated in the tissues, mainly because of the transstimulation capacity of system L to exchange LNAA (42); hence, BCAA are rapidly utilized. The metabolic fate of these amino acids in the mammary gland is to be incorporated into the newly synthesized milk proteins or to be transaminated by mBCAT, presumably producing an increase in intracellular glutamate concentration. The mammary tissue glutamate pool is considered the richest amino acid pool during lactation in various species (35). Milk proteins are rich in this amino acid, and it is also found as a free amino acid in maternal milk. The glutamate concentration in milk is higher compared with the rest of the free amino acids except glutamine and taurine (5, 6, 26). Glutamate and glutamine are important energy sources for the intestine (36) and are essential during the first days of life of the newborn. Transamination of BCAA also increases the production of BCKA, which in turn can be oxidatively decarboxylated by the BCKD (6), yielding energy or leading to de novo synthesis of fatty acids, as previously observed (45). Finally, but no less important, BCKA that are not oxidatively catabolized can be reaminated to their corresponding BCAA to sustain the accelerated rate of protein synthesis in the gland. The BCKA formed during transamination are readily reaminated with BCAA or glutamate as amino donors (41), and in vivo, up to 80% of BCKA formed may be reaminated and incorporated into proteins (18).

Studies of milk protein gene regulation have led to the identification of hormones, cis-acting elements, and transcription factors that control milk protein gene expression (29). The mechanism by which the BCAT gene is induced is unknown. However, in the mammary gland, mBCAT induction is possibly associated with the hormonal status present during late pregnancy and lactation. Lactogenesis takes place in two stages (11, 22). In stage I, a substantial increase in the number of alveoli occurs, and, toward the end of pregnancy, there is alveolar cell differentiation, which occurs during the last trimester of pregnancy in most species. Lactogenesis stage I begins between days 12 and 15 of pregnancy in the rat. Stage II occurs around parturition and leads to full lactation. During lactation stage II, increases in milk secretion, which depend on increased milk removal as lactation proceeds, may involve changes in both the number of alveolar cells and their activity (22, 39). mBCAT expression in the rat mammary gland followed the patterns established for lactogenesis stage I and lactation (Figs. 1-4). Interestingly, however, there was no increase during the transition from pregnancy to lactation, i.e., lactogenesis stage II. Several hormones are required for lactogenesis stage II in rats. Although prolactin and glucocorticoids are necessary, the abrupt decrease of progesterone near parturition is the trigger for the beginning of lactogenesis stage II (22). Prolactin is known to stimulate the induction of synthesis of milk proteins such as casein as well as lactose and milk fat (27). However, although despite alpha -, beta -, and gamma -caseins, alpha -lactalbumin, whey acidic protein, and transferrin are major milk proteins in the rat, only transferrin expression seems to be regulated under different hormonal control than the rest of the proteins (19, 24). This response is in part dependent on the cis-acting elements present in the promoter region of the genes of these proteins. In general, an abrupt increase in these proteins has been thought to be associated with parturition, although the problem has not been studied in sufficient detail.

There are a number of factors involved in the induction of genes in the mammary gland during lactation. Milk protein genes are induced from 10- to 100-fold by several transcription factors, including signal transducers and activators of transcription, nuclear factor I, Yin Yang 1, CAAT-enhancer binding protein (C/EBP), and glucocorticoid receptors (29). Some metabolic enzymes, codified by housekeeping genes such as carboxyl ester lipase (15) and acetyl-coenzyme A carboxylase (16), show more modest levels of induction. Regulation of the expression of this carboxylase is controlled by multiple promoters, resulting in different mRNA forms, but the mammary gland expresses only one of them (16), whereas the esterase is regulated by a member of the C/EBP family (CTFNF-1) (15). Some metabolic genes specific for lactating mammary gland are induced by various factors. For instance, beta -1,4-galactosyltransferase, involved in lactose biosynthesis, is expressed as a housekeeping transcript of 4.1 kb in somatic cells, whereas in the mammary gland it occurs as a transcript of 3.9 kb induced by specific transcription factors including AP2 (25, 33). Thus no single factor or signaling component is sufficient to determine the precise temporal and spatial patterns of gene expression in the mammary gland during lactation (29).

The factors that might regulate transcription of the mBCAT gene have not yet been determined. mBCAT exhibits near-ubiquitous expression in rat tissues; however, hormonal regulation of mBCAT expression has not been demonstrated conclusively outside the mammary gland (7). mBCAT does exhibit a modest increase in expression in skeletal muscle in response to increased dietary protein (40). On the other hand, mBCAT does show cell-specific expression in kidney, uterus, ovary, stomach, intestine, lung, and brain (37, 40). In many of these tissues, mBCAT is found in the secretory epithelial cells of the tissue (37). Thus studies on the regulation of the mBCAT gene will provide further insights into factors that regulate both cell-specific expression and developmental regulation of mammary gland genes. Mapping and functional assays of the promoter region of mBCAT will determine the transcriptional factors involved in the regulation of this gene during lactation. Interestingly, the results of this study showed that mBCAT expression does not change during lactogenesis stage II but does change with the increase in milk production, indicating that, perhaps, induction of mBCAT during this stage is not regulated hormonally but, rather, metabolically. Further research is needed to demonstrate this hypothesis.


    ACKNOWLEDGEMENTS

This work was supported by Grant 0592-M from the Consejo Nacional de Ciencia y Tecnología Mexico (to N. Torres), and by Grant DK-34738 from the National Institutes of Health (to S. M. Hutson)


    FOOTNOTES

Address for reprint requests and other correspondence: N. Torres, Departamento de Fisiología de la Nutrición, Instituto Nacional de Ciencias Médicas y Nutrición, Vasco de Quiroga 15, México City, 14000 Mexico, (E-mail: nimbet{at}quetzal.innsz.mx).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 6 June 2000; accepted in final form 31 October 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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