The Journal of Nutrition Vol. 128 No. 7 July 1998, pp. 1165-1171

Regulation of Branched-Chain Amino Acid Metabolism in the Lactating Rat^1,2

Soledad DeSantiago^*, Nimbe Torres^*, Agus Suryawan, Armando R. Tovar^{*, 3}, and Susan M. Hutson

^* Departamento de Fisiología de la Nutrición, Instituto Nacional de la Nutrición Salvador Zubirán, D. F. 14000, México and  Department of Biochemistry, Wake Forest University School of Medicine, Winston-Salem, NC 27157

	ABSTRACT

Abstract Introduction Methods Results Discussion References

There is evidence that during lactation, uptake of the essential branched-chain amino acids (BCAA) by mammary glands exceeds their output in milk protein. In this study, we have measured the potential of lactating rats to catabolize BCAA. The activity, relative protein and specific mRNA levels of the first two enzymes in the BCAA catabolic pathway, branched-chain aminotransferase (BCAT) and branched-chain -keto acid dehydrogenase (BCKD), were measured in mammary gland, liver and skeletal muscle obtained from rat dams at peak lactation (12 d), from rat dams 24 h after weaning at peak lactation and from age-matched virgin controls. Western analysis showed that the mitochondrial BCATm isoenzyme was found in mammary gland. Comparison of lactating and control rats revealed that tissue BCATm activity, protein and mRNA were at least 10-fold higher in mammary tissue during lactation. Values were 1.3- to 1.9-fold higher after 24 h of weaning. In mammary gland of lactating rats, the BCKD complex was fully active. In virgin controls and weaning dams, only about 20% of the complex was in the active state. Hypertrophy of the liver and mammary gland during lactation resulted in a 73% increase in total oxidative capacity in lactating rats. The results are consistent with increased expression of the BCATm gene in the mammary gland during lactation, whereas oxidation appears to be regulated primarily by changes in activity state (phosphorylation state) of BCKD.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

The first step in the catabolism of the nutritionally essential branched-chain amino acids (BCAA),⁴ leucine, valine and isoleucine, is reversible transamination catalyzed by the branched-chain aminotransferase (BCAT) (EC 2.6.1.42) to produce their respective branched-chain -keto acids. Two isoenzyme forms of BCAT are found in mammals, mitochondrial (BCATm) and cytosolic (BCATc) (Hutson et al. 1988 and 1992). The mitochondrial isoenzyme, BCATm, is found in most tissues, whereas BCATc is expressed only in brain, ovary and placenta (Hutson 1988, Hutson et al. 1988 and 1992, Ichihara 1985). In skeletal muscle and brain, BCAT is thought to play an important role in nitrogen metabolism (Gefland et al. 1986, Harper 1989, Hutson et al. 1978, Yudkoff et al. 1996). However, the precise function of BCAA transamination in other tissues has not been studied extensively.

The second and first irreversible step in the catabolic pathway is catalyzed by the mitochondrial branched-chain -keto acid dehydrogenase enzyme complex (BCKD) (EC 1.2.4.4). In this reaction, the branched-chain -keto acids are oxidatively decarboxylated to produce the corresponding branched-chain acyl-CoA derivatives. The mammalian BCKD complex contains multiple copies of three enzymes, a branched-chain -keto acid decarboxylase (E1) composed of 2 and 2 subunits, a dihydrolipoyl transacylase (E2) and a dihydrolipoyl dehydrogenase (E3) (Harris et al. 1986). The activity of the complex within a tissue is regulated by phosphorylation-dephosphorylation, catalyzed by a specific kinase (BCKDK) and phosphatase. The phosphorylation state of the complex is controlled primarily by the activity of the BCKD kinase (Fatania et al. 1981, Harris et al. 1986, Popov et al. 1992). As a result of the high BCAT activity in skeletal muscle and differences in activity state of the BCKD complex in skeletal muscle and liver, a complex metabolic scheme has evolved whereby BCAA transamination and oxidation appear to occur at different sites with substantial shuttling of BCAA metabolites between skeletal muscle and liver (Hutson et al. 1978, Shinnick and Harper 1976).

The validity of the above model, which was developed with the use of male rats, has yet to be established in female rats. Indeed, a recent study found gender-specific differences in the diurnal regulation of liver BCKD activity that were prevented by ovariectomy (Kobayashi et al. 1997). Although data on BCAA metabolism during pregnancy and lactation are limited, information that is available suggests that the lactating mammary gland may play a role in BCAA metabolism in the rat dams. At peak lactation, the dietary protein intake of dams is increased 100-200% (DeSantiago et al. 1991, Williamson 1980), but this is associated with increased turnover and a decrease in the concentration of leucine in blood (Viña and Williamson 1981). Blood flow and amino acid uptake into mammary gland during lactation are increased significantly (Trottier 1997, Viña et al. 1981a). In sows, BCAA uptake exceeds quantitative excretion in milk proteins (Trottier et al, 1997). During lactation, the rat mammary gland becomes a major site of leucine removal, and the lactating mammary gland has BCAT activity (Viña and Williamson 1981). Leucine catabolism has been reported in the lactating mammary gland (DeSantiago et al. 1998, Viña and Williamson 1981).

Our aim was to examine the effect of lactation on the potential of the mammary gland and tissues that are thought to be quantitatively important in body BCAA metabolism to catabolize the essential BCAA. Because regulation of BCAA catabolism is achieved by the tissue distribution and activity of the first two enzymes in the pathway (Hall et al. 1993a, Hutson et al. 1978, Shinnick and Harper 1976), we measured the effect of lactation and weaning on rat mammary gland, liver, skeletal muscle BCAT and BCKD activities, relative protein and specific mRNA levels.

	MATERIALS AND METHODS

Abstract Introduction Methods Results Discussion References

Animals.  Female Sprague-Dawley rats (220 g) (Instituto Nacional de Nutricion, Mexico, D. F.) were used. All animals were allowed free access to an 18% casein diet (Table 1; Rogers and Harper 1965) and water throughout the study.

The female rats were mated when they reached a body weight of 260 ± 20 g. Rats were caged individually after pregnancy was confirmed by the presence of spermatozoa in the vaginal smear. After normal pregnancy and delivery, the litter size was adjusted to eight pups per dam. The day of birth was considered as d 1 of lactation. Dams in the lactation group (n = 9) were killed during peak lactation at d 12 postpartum. In the weaning group (n = 9), pups were removed on d 12 postpartum; the dams were killed 24 h after removal of the pups. Virgin rats (n = 9), paired by age with the experimental groups, were used as the control group. The protocol complies with the National Institutes of Health Guide for the Use and Care of Laboratory Animals.

Tissue preparation and enzyme assays.  Rats were anesthetized with 30 mg/kg sodium pentobarbital before decapitation. Animals were killed between 0800 and 0930 h. All tissue used for enzyme assays and RNA preparations was removed rapidly and frozen immediately with the use of clamps precooled in liquid nitrogen. Frozen tissues were stored at 80°C. Liver, gastrocnemius muscle and mammary tissues were powdered with a porcelain mortar and pestle precooled with liquid nitrogen before homogenization and extraction. Protein was determined by the method of Lowry et al. (1951), with the use of crystalline bovine albumin as standard.

For the BCAT assays, frozen tissue was pulverized using a precooled mortar and pestle. Connective tissue was removed before the powder was transferred to cooled preweighed centrifuge tubes. The tissue was suspended in buffer (1 g tissue/3 mL extraction buffer) containing 25 mmol/L HEPES, pH 7.4, 4 g/L 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS), protease inhibitors (Hutson et al. 1995), 20 mmol/L EDTA, 10 mmol/L EGTA and 2 mmol/L dithiothreitol (DTT). The tissue suspension was then subjected to three rounds of freeze-thaw sonication (Hutson et al. 1995) before centrifugation at 12,000 × g for 10 min. The supernatant was assayed for BCAT activity at 37°C as described by Hutson et al. (1988) except that -keto[1-¹⁴C]isocaproate was used instead of -keto[1-¹⁴C]isovalerate. -Keto[1-¹⁴C]isocaproate was prepared from L-[1-¹⁴C]leucine (Dupont-NEN, Boston, MA) in accordance with the procedure of Rüdiger et al. (1972). A unit of enzyme activity was defined as 1 µmol of leucine formed per minute at 37°C.

Extraction of the BCKD complex from liver, skeletal muscle and mammary tissue was performed essentially as described by Block et al. (1987) and modified by Shimomura et al. (1993) with the use of 50-100 mg of tissue. BCKD activity was measured as ¹⁴CO₂ release from -keto[1-¹⁴C]isocaproate. Total BCKD complex activity, which is an estimate of enzyme amount, was measured after activation of a separate aliquot of the same sample in the presence of MgSO₄ and a broad specificity phosphatase as described in Shimomura et al. (1993). The phosphatase was purified from rat liver by using the protocol described in Harris et al. (1982). In preliminary experiments, we found that phosphatase treatment was required for full activation of the complex in mammary tissue extracts. The activity state of BCKD is the ratio of actual activity before activation to total activity obtained after activation by phosphatase treatment. One unit of BCKD complex catalyzed the production of 1 µmol of ¹⁴CO₂/min at 37°C.

SDS-PAGE and immunoblotting.  Proteins in the tissue extracts (50 µg) (n = 3 rats) were separated by SDS-PAGE in 10% gels according to Laemmli (1970). Before electrophoresis, all samples were boiled for 2 min in the presence of 1% SDS, with 5% -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 a nylon membrane (Hybond-N+, Amersham). Immunoblotting was performed as described by Hall et al. (1993) with the use of rat BCATm specific IgG fraction, E2 specific antiserum and E1 antiserum. The E2 and E1 antisera were the kind gift of Yoshi Shimomura, University of Nagoya, Japan. Immunoreactive protein bands were visualized using goat anti-rabbit immunoglogulin G conjugated with horseradish peroxidase-labeled goat anti-rabbit antibody according to the manufacturer's instructions (Bio-Rad, Hercules, CA). Densitometry was used to estimate relative protein levels.

RNA preparation and Northern analysis.  Total RNA was isolated from tissues (n = 5-6 rats) as described in Chomczynski and Sacchi (1987). For Northern blotting, 20 µg of total RNA was separated in an 0.8% agarose-formaldehyde gel and transferred to Hybond N+ membrane from Amersham (Buckinghamshire, UK). BCATm mRNA was detected using a partial rat BCATm cDNA probe (900 bp) (Bledsoe et al. 1997). The rat BCKD E2 cDNA probe was the gift of Robert A. Harris of the Indiana University School of Medicine, Indianapolis, IN. Polymerase chain reaction was used to obtain the E1 cDNA probe (1.2-kb fragment). The forward and reverse primers that were used, 5'-TCAGTATGGGCAACTCAGAA-3' and 5'-CATGTGCCTCTAAAGCGTTAC-3', respectively, were based on the sequence published by Zhang et al. (1991). The cDNA probes were labeled with 5'[-³²P]dCTP (111 MBq/mmol; Dupont-NEN) using a redi-prime DNA labeling system (Amersham). Membranes were prehybridized for 45 min in Rapid-Hyb buffer (Amersham) and hybridized with the radiolabeled probe (16.7 MBq/L) for 2.5 h at 65°C. Membranes were washed with 2X SSPE (1X SSPE = 150 mmol/L NaCl, 10 mmol/L NaH₂PO₄, 1 mmol/L EDTA) and 0.1% SDS (wt/v) at room temperature for 20 min followed by two 15-min washes in 0.1X SSPE plus 0.1% SDS at 65°C. Membranes were exposed to Ektascan film (Kodak de Mexico, Guadalajara, Mexico) at 70°C with an intensifying screen. An Instant Imager Electronic Autoradiography system (Amersham) was used to estimate message levels. Results are expressed as arbitrary units.

Statistical analysis.  The data were analyzed by one-way ANOVA, and significant differences were identified using Fisher's protected Least Significant Difference test (Stat View version 4.02, Abacus Concepts, Berkeley, CA). Differences were considered significant at P < 0.05.

	RESULTS

Abstract Introduction Methods Results Discussion References

At peak lactation (d 12), the energy and protein intakes of the rat dams were 1.7-fold higher than in the age-matched virgin control rats. In the weaning group, from which pups were removed on d 12 of lactation, energy and protein intakes were still 1.1-fold higher than in the control virgin rats and only 21% lower than in the lactation group. When energy and protein intakes were adjusted for the differences in body weights, energy (Table 2) and protein intakes (not shown) were still 1.5-fold and 90% higher in the lactating and weaning groups, respectively, than in the virgin control group. Hypertrophy of the mammary gland (5 g in control vs. 13.9 g in lactation group) accompanied lactation. The mammary gland in the weaning group was engorged with milk and was double the wet weight of the gland in the lactation group. The mammary gland of the lactating dams and weaning group had a greater protein concentration than that of the virgin controls (Table 2).

Hypertrophy of the liver was observed during lactation and weaning, but liver protein concentration did not differ among the three groups. There were no significant differences in skeletal muscle weight or protein concentration in control virgin rats, lactating dams and dams 24 h after weaning.

BCAT activity, protein and mRNA levels.  The effect of lactation and weaning on the activity of the first enzyme in the BCAA catabolic pathway, BCAT, was examined in mammary gland and skeletal muscle from control, lactating and weaning rats (Table 3). BCAT activity in mammary tissue from lactating rats was 10 times that of control rats when expressed as units per gram wet tissue and 4.2 times that of control rats when specific activities (mU/mg protein) were calculated. Twenty-four hours after the pups were removed, mammary gland BCAT activity was lower than in lactating dams, but BCAT activity per gram of wet tissue was still 1.9-fold higher than in virgin controls. In skeletal muscle, although activity differences between the weaning and control groups (units/g wet tissue) were significant (48% higher in weaning rat skeletal muscle than in control rats, P < 0.05), specific activities did not differ among the three groups (Table 3).

When the differences in tissue weight were taken into account, the total capacity of the mammary tissue to transaminate BCAA was 27-fold higher during lactation (Table 3). Skeletal muscle capacity did not differ among the three groups. Nevertheless, assuming that the leg gastrocnemius muscle is representative of bulk skeletal muscle, during lactation, skeletal muscle capacity was preserved in the face of the large increase in transamination capacity in the mammary gland.

As shown in Figure 1 (panel A), BCATm is the BCAT isoenzyme present in the mammary gland. The cytosolic isoenzyme, BCATc, was not present in mammary tissue (data not shown). There was a lactation-dependent change in BCATm isoenzyme protein content in mammary tissue (Fig. 1, panel A). When equal amounts of protein were loaded on the gel, eightfold differences in the relative amount of BCATm protein in lactating mammary gland (31.7 ± 2.42 units) and in virgin gland (3.48 ± 0.16 units) were found (P < 0.01). Values for the weaning group were 1.3-fold higher than in controls (P = 0.25). Differences in levels of BCATm protein were similar to differences in activity. Lactating, weaning and control rat skeletal muscle BCATm protein levels were not significantly different.

View larger version (71K): [in this window] [in a new window]   Fig 1. Effect of lactation and weaning on mitochondrial branched-chain aminotransferase (BCATm) protein (panel A) and mRNA (panel B) levels in rat skeletal muscle, mammary tissue and liver. Tissues from virgin control rats (lanes 1 and 4), lactating rats (lanes 2 and 5) and weaning rats (lanes 3 and 6) were used for the analyses. Panel A: Immunoblotting. Frozen tissue (500 mg) was homogenized and extracted as described in Materials and Methods. Supernatant protein (50 µg) from each tissue was subjected to 10% SDS-PAGE, transferred to a nitrocellulose membrane and immunoblotted using rat anti-BCATm-specific immunoglobulin G as described in Materials and Methods. Panel B: Northern blot hybridization. Total RNA (20 µg) from tissues from the same rats as in panel A was subjected to electrophoresis, blotted, and hybridized with the uniformly 32P-labeled BCATm cDNA probe as described in Materials and Methods. Panel C: Ethidium bromide staining of the gels used for Northern blot hybridization in panel B.

Northern-blot analysis was used to estimate levels of BCATm mRNA in mammary tissue and skeletal muscle of control virgin, lactating rats and in rats 24 h after weaning (Fig. 1, panel B). The quantities of RNA subjected to electrophoresis were comparable as indicated by ethidium bromide staining of the ribosomal RNA bands. The level of BCATm mRNA was 4.9-fold higher (n = 6, P < 0.001) in the mammary tissue of lactating rats than in controls. After 24 h of weaning, mRNA levels had decreased nearly to the level found in virgin mammary tissue (60% higher than control levels). As suggested by the activity data, differences in skeletal muscle BCATm mRNA among the three groups were small, and only the difference between weaning and control groups was significant (4.1 ± 1.3 vs. 9.5 ± 1.5 units, P < 0.05).

BCKD activity, subunit protein and mRNA levels.  Because the results suggested a lactation-dependent increase in expression of BCATm, it was of interest to determine whether BCKD activity (oxidative capacity) and expression were also affected by lactation. During lactation, mammary tissue actual BCKD activity (mU/g wet tissue) was 17-fold higher than in control rat mammary gland (Table 4). In contrast to virgin rat mammary gland, in which only 22% of the BCKD was in the active form, essentially all of the enzyme was in the active form in the lactating mammary gland. After phosphatase treatment, total mammary gland BCKD activity (mU/g wet tissue) was about threefold greater in lactating rats than in controls (Table 4). When specific activity was calculated, actual activity in the tissue extract was about fourfold higher, whereas total activity in proportion to tissue protein did not differ (data not shown). Within 24 h after weaning, BCKD activity and activity state were not different from control values. On the other hand, actual and total BCKD activities in liver did not differ among groups. In skeletal muscle, a significant difference was found only in actual BCKD activity between controls and lactating rats (Table 4).

View this table: [in this window] [in a new window]   Table 4. Branched chain -keto acid dehydrogenase (BCKD) activity in mammary gland, liver and skeletal muscle from virgin control, lactating and weaning rats1

Calculations of oxidative capacity based on total organ and tissue weights in the three groups revealed that actual oxidative capacity in the mammary gland was 50-fold higher and total tissue capacity was ninefold higher in the lactating group than in the controls (Table 4). Because of the liver hypertrophy during lactation and at weaning (24 h), liver oxidative capacity, i.e., actual and total liver BCKD activity were also significantly greater than in control virgin rats. When combined, the changes in liver and mammary tissue BCKD activity that occurred during lactation resulted in a 73% higher capacity of lactating rats to oxidize BCAA than virgin control rats. Although mammary tissue actual and total activities were less than in lactating rats within 24 h after weaning, liver capacity remained elevated as a result of the liver hypertrophy; thus no net decrease in total oxidative capacity occurred.

The effect of lactation and weaning on levels of tissue BCKD subunit proteins was investigated by using Western blot analysis with E2 and E1 antisera to determine whether levels of the BCKD complex protein subunits were affected by lactation and weaning (Fig. 2, panels A and B). Northern blot analysis was used to quantify E2 and E1 subunit mRNA levels (Fig. 2, panels C and D). Estimates of the levels of E2 protein revealed 2.3-fold higher levels of E2 protein in mammary tissue during lactation than in glands of contol virgin rats (P < 0.05). By 24 h after weaning, E2 protein had returned to control levels. Differences in relative levels of E2 mRNA in control and lactating rats gland, estimated using the Instant Imager (Fig. 2D), were even greater than differences in protein levels (n = 5 rats per group, P < 0.001). Relative amounts of E2 mRNA in lactating, weaning and control groups were 42.7 ± 1.0, 24.8 ± 3.9 and 2.8 ± 0.9 units, respectively. Western blot analysis with the E1 antiserum, which detects the E1, E1 and E2 proteins (Hisao et al. 1995), also showed higher levels of E2 protein in the mammary gland of lacatating rats than in control rats (Fig. 2B). No significant differences in the E1 and E1 protein levels were detected among groups. Levels of E1 mRNA were only 60% higher in the lactating rats than in control virgin rats (P < 0.05, n = 6). Thus differences in E2 mRNA between control and lactating groups were consistent with the higher observed levels of E2 protein in the lactating mammary gland. However, comparable changes in E1 and E1 protein and mRNA were not observed (Fig. 2C, D).

View larger version (82K): [in this window] [in a new window]   Fig 2. Effect of lactation and weaning on levels of branched-chain -keto acid dehydrogenase (BCKD) subunit proteins (panels A and B) and mRNA (panels C and D) in rat skeletal muscle, mammary tissue, and liver. The following order is used under each tissue heading: control tissue is in the left lane, lactating rat tissue in the middle lane and weaning rat tissue in the right lane. Panel A and B: immunoblotting. Frozen tissue (500 mg) was homogenized and extracted as described in Shimomura et al. (1993). Extracted protein (50 µg) from each tissue was subjected to 10% SDS-PAGE, transferred to a nitrocellulose membrane and immunoblotted using anti-E2 antiserum (panel A) or anti-E1 antiserum (panel B) as described in Materials and Methods. Panels C and D: Northern blot hybridization. Total RNA (20 µg) from tissues from the same rats as in panels A and B was subjected to electrophoresis, blotted and hybridized with the uniformly 32P-labeled E1 cDNA probe (panel C) or E2 cDNA probe (panel D) as described in Materials and Methods. Panel E: ethidium bromide staining of the gels used for Northern blot hybridization in panels C and D.

Differences in E2 protein and mRNA levels in skeletal muscle among groups were not significant and were not coordinated with complex activity during lactation and weaning. In liver, E2 mRNA did not differ among groups. No effect of lactation or weaning on E2 protein levels was observed. Levels of E1 mRNA in liver did not differ among the three groups. Levels of E1 protein were significantly higher in the lactating rat liver than in controls, but E1 protein levels did not differ.

	DISCUSSION

Abstract Introduction Methods Results Discussion References

We examined the effect of lactation on the enzymes that control the tissue-specific catabolism of the essential BCAA, BCAT and the BCKD complex. We have demonstrated for the first time that BCATm is the isoenzyme expressed in the mammary gland and have presented evidence suggesting regulation of expression of the gene for BCATm. We have found that lactation results in activation of the BCKD in mammary tissue, which was accompanied by higher levels of the mRNA and protein for the E2 enzyme subunit of the BCKD complex. The sum of the changes in BCATm and BCKD activities in mammary gland and liver, and the generally unchanged oxidative capacity in skeletal muscle, result in a greater capacity of lactating rat dams to oxidize essential BCAA compared with virgin rats.

Our results are consistent with the hypothesis that the hormonally mediated changes in the mammary gland that occur during pregnancy and lactation result in increased expression of the BCATm gene. On d 12 of lactation when tissue samples were taken, profound changes had occurred in the RNA content and protein content of the gland compared with the virgin controls (DeSantiago et al. 1991). The RNA content was 8.55 mg/g at peak lactation compared with 0.5 mg/g in age-matched controls (DeSantiago et al. 1991). As a result, the 4.9-fold higher level of BCATm-specific mRNA found in the lactating gland than in the virgin gland actually represents about a 100-fold increase in the concentration of BCATm message in the tissue. The measured protein content of the mammary gland per gram tissue was 4.8-fold higher during lactation (Table 2), suggesting that the eightfold increase in BCATm protein was 50-fold increase in BCATm concentration in the gland. Therefore the differences in BCATm mRNA, protein and activity (Table 3 and Fig. 1) are consistent with a pregnancy/lactation induction of BCATm gene expression. Our estimate of the half-life of the BCATm mRNA (>24 h) (Tovar, R. A., of The National Institute of Nutrition, Mexico, unpublished results) makes it unlikely that a change in mRNA stability during pregnancy or lactation is the mechanism responsible for the increase in BCATm expression because of the change in BCATm mRNA half-life that would be required to effect a 10- to 100-fold increase in message levels. On the other hand, the cell type(s) that expresses BCATm in the mammay gland is not yet known, and the mammary gland changes dramatically in cell population during pregnancy and lacatation (Hobbs et al. 1982, Munford 1963, Pitelka 1988). Thus it is possible that the changes in expression of the BCATm gene reflect changes in gene transcription, increased proliferation of a specific-cell population or both mechanisms.

Irreversible loss of the essential BCAA carbon skeleton occurs at the BCKD step. The E2 subunit was found in higher concentration in the mammary gland of lactating rats, but levels of the other subunits did not differ significantly. Therefore, there was no coordinated change in the levels of BCKD complex subunit enzyme proteins. Oxidative capacity increased dramatically because more of the complex was in the active state (Table 4). The oxidative capacity of the gland increased because of hypertrophy of the tissue.

The change in activity state of the gland from 20% to essentially 100% active is likely to have resulted from changes in the level of BCKD kinase activity in the lactating mammary gland. There is a considerable body of evidence to support the current hypothesis that the activity state of the BCKD complex is regulated primarily by the activity of its kinase (Fatania et al. 1981, Harris et al. 1986, Popov et al. 1992). Liver, which has the highest activity state of any other tissue in male rats, contains the lowest amount of kinase protein and mRNA (Popov et al. 1995). It has also been reported that female hormones regulate kinase activity and/or expression in rat liver. Kobayashi et al. (1997) found an increased association of the BCKD kinase with the BCKD complex, increased kinase activity and inactivation of the BCKD complex at the end of the light cycle in female, but not in male rats. Ovariectomy prevented the diurnal inactivation of the complex. Although preliminary, our results suggest that regulation of BCAA oxidation in the mammary gland during lactation may involve alterations in kinase levels and/or its association with the BCKD complex. The importance of the change in E2 subunit levels in the gland is not yet understood.

Although the tissue-specific metabolic adaptations in fat and carbohydrate metabolism that occur in lactating rats to meet the energy requirements for lactation are well known (reviewed in Williamson 1980), there is much less information on tissue-specific responses in protein metabolism during lactation. As discussed by Trottier (1997), amino acid requirements of the mammary gland itself and the fate of retained amino acids have not been investigated. Previous studies in rats (Viña et al. 1981a, 1981b and 1981c), dairy cows (Laarveld et al. 1981) and sows (Trottier et al. 1997) have shown a channeling of BCAA from the blood pool to the mammary tissue. In the bovine mammary gland, extraction of BCAA accounts for 53% of total amino acids extracted by this tissue (Laarveld et al. 1981). Viña and Williamson (1981) reported a 35% increase in leucine turnover in lactating rats and demonstrated that mammary gland explants oxidize leucine in vitro. Estimates of leucine and valine oxidation by mammary gland from several species including rats are on the order of 25-30% of amino acid taken up by the gland (Davis and Mephram 1976, Roets et al. 1979, Viña and Williamson 1981, Wohlt et al. 1977).

Our results show that the capacity of the mammary gland to catabolize BCAA increases dramatically during lactation. Liver oxidative capacity increases largely as a result of liver hypertrophy rather than changes in BCKD activity. Skeletal muscle transaminase and BCKD levels did not change dramatically. The high rate of blood flow to the mammary gland during lactation and retention of BCAA in excess of the requirement for milk production (Trottier 1997) would permit a shift in oxidation from liver to the gland. Unlike the liver, which must extract branched-chain -keto acids from the blood, the mammary gland contains BCATm. If both BCATm and BCKD are expressed in the same cell type in the mammary gland, direct channeling of -keto acids produced by transamination to the site of oxidation is possible. At weaning, food intake of the dams was still elevated, and our results show that the liver is poised to handle the excess BCAA. However, transamination still must occur extrahepatically. It is not known whether mammary tissue provides branched-chain -keto acids for hepatic oxidation.

The high ratio of BCAT to actual BCKD activity in the mammary gland also raises questions about the function of transamination in the gland (Tables 3, 4). The upregulation of catabolic enzymes in a tissue in which the primary function is anabolic (i.e., to synthesize milk proteins) suggests a role for these amino acids in addition to their use as protein precursors. Furthermore, as discussed by Richert et al. (1996), it is difficult to attribute the large improvement in litter weaning weights in response to small additions of valine to the sow's diet solely to the use of valine as an energy source. It has been hypothesized that in skeletal muscle, BCAA transamination provides nitrogen for synthesis of glutamate and glutamine (reviewed in Harper 1989); recently, we found that leucine stimulates glutamine efflux in primary astrocyte cultures (Hutson, S, M., of The Wake Forest University, NC, unpublished results). In addition to the amino acids found in milk proteins, rat milk contains high concentrations of glutamate and glutamine (Davis et al. 1993). A strong positive correlation between plasma glutamate plus glutamine concentration and rates of amino acid oxidation in humans has also been reported (Motil et al. 1994). The mammary gland may also participate in the interorgan shuttling of BCAA metabolites and in the metabolism of glutamate and glutamine as has been postulated for skeletal muscle. Clearly, more work is required to understand the physiologic significance of the changes in BCAA pathway enzymes and interorgan partitioning of BCAA metabolism during pregnancy and lactation.

	FOOTNOTES

Manuscript received 14 November 1997. Initial reviews completed 7 January 1998. Revision accepted 18 February 1998.

	ACKNOWLEDGMENT

The authors wish to acknowledge Dora Il'yasova for technical assistance.

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