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Am J Physiol Endocrinol Metab 285: E854-E863, 2003. First published June 17, 2003; doi:10.1152/ajpendo.00153.2003
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Potential role of leucine metabolism in the leucine-signaling pathway involving mTOR

Christopher J. Lynch,1 Beth Halle,1 Hisao Fujii,2 Thomas C. Vary,1 Reidar Wallin,3 Zahi Damuni,1 and Susan M. Hutson2

1Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033; and Departments of 2Biochemistry and 3Internal Medicine, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157

Submitted 8 April 2003 ; accepted in final form 10 June 2003


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Leucine has been shown to stimulate adipose tissue protein synthesis in vivo as well as leptin secretion, protein synthesis, hyper-plastic growth, and tissue morphogenesis in in vitro experiments using freshly isolated adipocytes. Recently, others have proposed that leucine oxidation in the mitochondria may be required to activate the mammalian target of rapamycin (mTOR), the cytosolic Ser/Thr protein kinase that appears to mediate some of these effects. The first irreversible and rate-limiting step in leucine oxidation is catalyzed by the branched-chain {alpha}-keto acid dehydrogenase (BCKD) complex. The activity of this complex is regulated acutely by phosphorylation of the E1{alpha}-subunit at Ser293 (S293), which inactivates the complex. Because the {alpha}-keto acid of leucine regulates the activity of BCKD kinase, it has been suggested as a potential target for leucine regulation of mTOR. To study the regulation of BCKD phosphorylation and its potential link to mTOR activation, a phosphopeptide-specific antibody recognizing this site was developed and characterized. Phospho-S293 (pS293) immunoreactivity in liver corresponded closely to diet-induced changes in BCKD activity state. Immunoreactivity was also increased in TREMK-4 cells after the induction of BCKD kinase by a drug-inducible promoter. BCKD S293 phosphorylations in adipose tissue and gastrocnemius (which is mostly inactive in vivo) were similar. This suggests that BCKD complex in epididymal adipose tissue from food-deprived rats is mostly inactive (unable to oxidize leucine), as is the case in muscle. To begin to test the leucine oxidation hypothesis of mTOR activation, the dose-dependent effects of orally administered leucine on acute activation of S6K1 (an mTOR substrate) and BCKD were compared using the pS293 antibodies. Increasing doses of leucine directly correlated with increases in plasma leucine concentration. Phosphorylation of S6K1 (Thr389, the phosphorylation site leading to activation) in adipose tissue was maximal at a dose of leucine that increased plasma leucine approximately threefold. Changes in BCKD phosphorylation state required higher plasma leucine concentrations. The results seem more consistent with a role for BCKD and BCKD kinase in the activation of leucine metabolism/oxidation than in the activation of the leucine signal to mTOR.

mammalian target of rapamycin; ribosomal protein S6 kinase-1; branched-chain {alpha}-keto acid dehydrogenase

IN ADDITION TO ITS ROLE as an insulin secretagogue, leucine appears to be a nutrient signal that regulates protein synthesis in adipose as well as other tissues by mechanisms that are independent of insulin (45). This effect appears to be the result of a direct action of leucine or a leucine metabolite on the tissue, since it can be reproduced in isolated cells. In addition, these effects are mimicked in vitro and in vivo by the leucine analog norleucine, which is not an insulin secretagogue (43–45). The effects of leucine on protein synthesis are brought about, at least in part, by activation of a cell-signaling pathway involving the mammalian target of rapamycin (mTOR), a serine/threonine protein kinase. In adipose tissue, the mTOR-signaling pathway appears to play an essential role in protein synthesis, differentiation of preadipocytes, adipose tissue morphogenesis, hypertrophic growth, and leptin secretion (for review see Ref. 41). In particular, a substrate of mTOR, eukaryotic initiation factor 4E-binding protein-1 (4E-BP1), appears to be a novel regulator of adipogenesis and metabolism (63). It is posited that leucine, through its regulation of mTOR signaling, also regulates these functions. For example, it has been shown that leucine regulates the organization of adipocytes into tissue-like structures (14), adipose tissue leptin synthesis/secretion (57), and protein synthesis (44, 45).

Leucine stimulates protein synthesis by activation of intracellular cell-signaling pathways such as those used by insulin. These are regulated by adapter proteins, such as RAPTOR (22, 31), and by phosphorylation/dephosphorylation cascades [e.g., mTOR to ribosomal protein S6 kinase-1 (S6K1) to S6] that, in turn, regulate factors involved in protein synthesis initiation and, to some extent, peptide chain elongation (7, 13, 21, 29, 33, 35, 54, 56, 64). It has been observed that inhibition of mTOR by rapamycin is only partially effective in blocking some effects of leucine or amino acids (3, 34, 61), suggesting that rapamycin-sensitive and -insensitive pathways could be involved. The rapamycin-sensitive pathway is thought to involve mTOR; however, less is known about the rapamycin-insensitive pathways. Activation of the serine/threonine protein kinase activity of mTOR leads to phosphorylation of several substrates, including S6K1 and the translational repressor 4E-BP1. The role of these proteins in protein synthesis regulation has been reviewed extensively (4, 20, 30, 32, 39, 40, 48, 55).

It is presently unclear exactly how leucine activates mTOR aside from the fact that the mechanism differs from that used by insulin. No leucine "receptor" has yet been identified. In fact, it has not been unequivocally established whether these effects of leucine are mediated by leucine or a metabolite of leucine such as {alpha}-ketoisocaproate (KIC) or the process of leucine oxidation itself (e.g., compare Refs. 29, 43, 46, 49, 62, and 65). A limiting factor in this regard has been that specific inhibitors of the first reversible step in leucine metabolism that produces KIC are not available. The commonly used inhibitors, such as aminooxyacetic acid (43, 46, 49, 62, 65), are nonspecific and inhibit a number of transaminases in the cell (28, 38) in addition to the one of interest, mitochondrial branched-chain amino acid (BCAA) transaminase (BCATm; Fig. 1). This complicates the interpretation of results obtained with such compounds.



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  		Fig. 1. First 2 steps in mitochondrial metabolism of leucine and alignment of the deduced amino acid sequences from the rat and human branched-chain {alpha}-keto acid dehydrogenase (BCKD) E1{alpha} subunit surrounding phosphorylation sites 1 and 2. A: schematic representation of the first 2 steps in leucine metabolism. The first step is rapid and reversible and, in most peripheral tissues, is catalyzed by the mitochondrial isoform branched-chain aminotransferase (BCATm). The second step catalyzed by BCKD complex is irreversible and regulated by BCKD kinase. "Lip" indicates lipoyl domains. B: alignment of rat and human sequences of the E1{alpha} subunit around the 2 phosphorylation sites, sites 1 and 2. Site 1 is Ser293 (S293), using numbering from Harris et al. (66) of mature rat protein sequence (i.e., mitochondrial import sequence removed).

 

Xu et al. (65) proposed a mechanism to explain how leucine stimulates mTOR signaling. Their hypothesis is that a metabolically linked signal arising from activation of leucine metabolism in the mitochondria results in mTOR activation. A potential target cited was the branched-chain {alpha}-keto acid dehydrogenase (BCKD) complex or its kinase.

The BCKD multienzyme complex catalyzes the rate-limiting and first irreversible step in leucine oxidation. The BCKD complex oxidatively decarboxylates KIC to form isovaleryl-CoA and NADH (Fig. 1). The complex contains a branched-chain keto acid decarboxylase (E1, which has an {alpha}2{beta}2 structure with a covalently bound thiamine pyrophosphate cofactor), dihydrolipoyl transacylase (E2, which has a covalently bound lipoic acid), and the flavin-linked dihydrolipoyl dehydrogenase (E3) (for review see Ref. 23). The {alpha}-keto acid of leucine, KIC, is thought to inhibit the BCKD kinase, promoting activation of BCKD complex. BCKD kinase phosphorylates the E1{alpha} subunit of BCKD at two sites, termed sites 1 and 2 (26, 51). Site-directed mutagenesis studies (66) have shown that phosphorylation at site 1 [Ser293 (S293)] inhibits BCKD activity, whereas phosphorylation at site 2 is silent.

The metabolic hypothesis from McDaniel's group (46, 65) is supported by several observations in addition to studies employing aminooxyacetic acid. First, at least two groups have reported that KIC is more efficacious than leucine at activating mTOR signaling (49, 65). Second, the order of potency of leucine, isoleucine, and valine and the importance of leucine in the activation of mTOR signaling in adipocytes and gastrocnemius (2, 43) are similar to the ability of their respective keto acids to inhibit BCKD kinase (17, 50). Third, a significant fraction of the mTOR in living cells appears to be associated with mitochondria (11), which would put it in an excellent position to be regulated by a mitochondrial signal. Last, on the basis of in vitro findings, adipose BCKD activity has been proposed as a potential target for direct regulation by changes in plasma leucine, although this has never been demonstrated in vivo (15). The implication of the Xu et al. (65) hypothesis is that a step in leucine metabolism provides both the signal transduction mechanism and the removal through metabolism of the leucine-KIC signal through one activity.

To begin to evaluate this hypothesis, we initiated studies to compare the effects of leucine on BCKD activity and S6K1. Because epididymal adipose tissue is limiting, we developed an antibody that allows us to measure BCKD phosphorylation state at site 1. In this study, we have evaluated this new antibody and have begun to use it to explore the role of BCKD regulation in mTOR activation. Our results show that adipose BCKD activity and phosphorylation can be regulated by leucine intake, as is the case for mTOR. The concentration dependence of the response suggests that the activation of adipose BCKD in response to rises in plasma leucine is more likely involved in regulating the metabolism of leucine than in the activation of mTOR signaling.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Animals and materials. The animal protocols were approved by the Penn State College of Medicine Institutional Animal Care and Use Committee. Male Sprague-Dawley rats (75–125 g) were purchased from Charles River Laboratories (Cambridge, MA) and maintained for >=7 days before the start of the treatment protocol on Teklad rat chow (24% protein) and water ad libitum. In some experiments, animals were then maintained for 7 days on Teklad diets containing 8, 24, or 50% protein (27). Subsequently, the livers were freeze-clamped and powdered under liquid nitrogen.

Enzyme purification. BCKD complex (10) and protein phosphatase 2A (PP2A) (1) were purified from bovine kidney as previously described.

Western blotting and antibodies. Phospho-[C-YRIGHH-(phospho-Ser)-TSDDSS] and unphosphorylated (C-YRIGHH-Ser-TSDDSS) peptides corresponding to phosphorylation site 1 on the E1{alpha} subunit of BCKD E1 were synthesized with a COOH-terminal cysteine residue to allow cross-linking to keyhole limpet hemocyanin. The peptides were purified by HPLC, verified by mass spectrometry, and lyophilized. The phosphorylated peptide was cross-linked to keyhole limpet hemocyanin and the conjugate used to prepare antiserum in rabbits, as previously described (42). The peptides were cross-linked to agarose with cyanogen bromide. The cross-linked beads were used to sequentially purify antibodies reacting with the peptide and the subset of peptide-specific antibodies that recognized the phosphopeptide alone. The phosphospecific IgGs were stored at 0.7–0.8 mg/ml in Tris-citrate-phosphate buffer, pH 7–8, with 0.1% sodium azide (NaN3).

The BCKD E1 antibody, used to evaluate loading of the gels, was generated against E1 of the purified rat liver BCKD complex as has been used previously to identify BCKD subunits in primary rat brain cell cultures and other tissues by Western blot analysis (28, 44). E1 and E2 antisera were obtained from Dr. Yoshi Shimomura (Nagoya Institute of Technology, Nagoya, Japan) and affinity purified using purified rat liver BCKD complex as described previously (44). BCKD kinase antibodies were raised in rabbits against purified recombinant BCKD kinase and were affinity purified. Duplicate gels were run using the same liver extract. One blot was first probed with the pS293 BCKD antibody, and subsequently the membrane was stripped according to the manufacturer's protocol and reprobed with antibodies that detect (a) total E1{alpha}. The duplicate blot was first probed with the E2 antibody and then with the BCKD kinase antibody. After this, the membranes were stained with Coomassie blue to verify protein loading. Anti-active [phospho-Thr389 (pT389)] S6K1 and total S6K1 ratios were determined as previously described (45).

Immunoreactivity on Westerns blots made using the aforementioned antibodies was quantified initially by densitometry after autoradiography and independently assessed using a GeneGnome quantitative enhanced chemiluminescence detection system from Syngene (www.syngene.com/tech.asp). X-ray films were made of each blot for a permanent record.

BCKD complex activity. Extraction of the BCKD complex from tissues (100 mg of tissue) was performed essentially as described by Shimomura and colleagues (58, 59). BCKD activity was measured by NADH appearance using {alpha}-ketoisovalerate as the keto acid substrate. Total BCKD complex activity was measured after activation of a separate aliquot of the same sample in the presence of 2 mM MnCl and PP2A or {lambda}-phosphatase. The activity state of BCKD is the ratio of actual activity before activation to total activity obtained after activation by phosphatase treatment. A unit of activity is defined as 1 µmol NADH formed per minute at 37°C.

Acute oral leucine administration. Animals were food deprived for 18 h and then allocated to one of the following groups (4–6 per group): saline(control) or leucine-administered group. At time 0, the animals were administered orally (by gavage) phosphate-buffered saline (control) or a dose of L-leucine (0.27, 0.54, 1.35, 2.7, or 5.4 g/dl) using a dose volume of 2.5 ml/100 g body wt (i.e., 6.75, 13.5, 33.2, 67.5, or 135 mg/100 g body wt). The highest dose of leucine is equivalent to the amount of leucine consumed by rats of this age and strain during 24 h of free access to a commercial diet (18). In one experiment, the average weight of the animals was 156 ± 5 g, and in another the average weight was 93 ± 1 g.

TREMK-4 cells. TREMK-4 cells, which contain a recombinant BCKD kinase gene under the control of a drug-inducible promoter, were a generous gift from Dr. Dean Danner (Emory School of Medicine, Atlanta, GA). The cells were maintained in cell culture, and BCKD kinase was induced as described previously (12). To examine phosphorylation end points, the cells were washed once and then incubated for 1 h with Krebs-Ringer-HEPES buffer (KRH) at 37°C. The medium was removed and replaced for 30 min with either fresh KRH or KRH with 4x amino acids [where 1x represents the amino acid concentration found in the postabsorptive state (52)].


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 DISCLOSURES
 REFERENCES
 
Characterization of pS293 E1{alpha} antibody. Figure 1 shows the first two steps in leucine metabolism that are present in adipose tissue (BCATm and BCKD) as well as the human and rat sequences that include phosphorylation site 1 and site 2 on the E1{alpha} subunit of BCKD. Phosphopeptide-specific antibodies to a phosphopeptide-encompassing site 1 on the E1{alpha} subunit of BCKD were affinity purified from rabbit serum. ELISA analysis of the affinity-purified phosphorylation site-specific antibody using the phosphopeptide (dilution titer 1:144,850) and unphosphorylated peptide (dilution titer 1:570) as antigens indicated that the purified antibody selectively recognized the phosphopeptide with a reactivity ratio of >99.5:1.

Additional characterization was performed to help evaluate the specificity of the antibody for pS293 on the E1{alpha} subunit that is responsible for BCKD inactivation. Purified BCKD complex from bovine kidney was incubated with excess ATP until the E1{alpha} subunit was phosphorylated extensively by its associated kinase (P lanes in Fig. 2). The extent of phosphorylation was determined on the basis of correlative [{gamma}-32P]ATP incorporation experiments (not shown). PP2A was also purified from kidney (1), and another sample of BCKD complex was incubated with activated PP2A to dephosphorylate the BCKD complex (D lanes in Fig. 2). Aliquots of these preparations at the indicated protein concentrations were separated by SDS-PAGE and transferred to PVDF membranes. The blots were then probed with pS293 BCKD antiserum. The results indicate that the antibody is highly selective for the phosphorylated form of BCKD (Fig. 2). Furthermore, pS293 immunoreactivity increased with increasing amounts of the phosphorylated BCKD loaded on the gels.



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  		Fig. 2. BCKD E1{alpha} site 1 phosphopeptide antibody immunoreactivity toward purified phosphorylated and dephosphorylated BCKD. Bovine kidney BCKD complex was phosphorylated (P) or dephosphorylated (D) by incubation with ATP or protein phosphatase 2A (10 U), respectively. An aliquot of assay mixture was solubilized in SDS-PAGE sample buffer. Different amounts of BCKD were subjected to SDS-PAGE and immunoblotting as described in EXPERIMENTAL PROCEDURES.

 

We next characterized the antibody in more complex samples. Harris et al. (27) have previously shown that amounts of hepatic BCKD protein and activity are influenced by the amount of protein in the diet. By use of their protocol, rats were fed diets containing 8, 24, or 50% protein for 7 days. Polyethylene glycol (PEG) precipitates of the frozen liver extracts were prepared, and the same precipitates were used to measure BCKD activity and for Western blotting (see Table 1 and Fig. 3). Consistent with previous findings (27), the liver enzyme was largely in the active state (92%) in animals fed a standard Teklad rat chow diet (24% protein). Increasing the protein content of the diet to 50% led to an increase in total BCKD activity, and essentially all of the BCKD was in the active state (99%). Livers from rats fed the low-protein diet had both lower activity state and lower total BCKD activity compared with livers from animals fed the standard or high protein diet.


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  		Table 1. Effect of dietary protein content on BCKD activity and E1{alpha} phosphorylation at site 1 in rat liver

 


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  		Fig. 3. Effect of dietary protein on hepatic BCKD and BCKD kinase and phosphorylation of BCKD on pS293. Liver proteins in the polyethylene glycol (PEG) pellet (18 µg) were separated by SDS-PAGE and transferred to PVDF membranes. Blots were probed with either immunoaffinity-purified BCKD E1 subunit antibodies, BCKD E2 subunit antibodies, pS293-specific antibodies, or antibodies to recombinant BCKD kinase, as indicated. The membrane was stained with Coomassie blue to verify protein loading (top).

 

As shown in Fig. 3, changing the protein content of the diet affected concentrations of BCKD enzyme subunits, BCKD kinase, and E1{alpha} phosphorylation. E2 and E1 concentrations increased as dietary protein was raised from 8 to 50%. On the other hand, BCKD kinase levels decreased, paralleling the changes in activity state, and were at the limits of detection in rats fed the 50% protein diet. The pS293 immunoreactivity also decreased with increasing protein in the diet. Because the dietary changes resulted in changes in E1{alpha} concentration, we examined the ratio of pS293 to E1{alpha} (total BCKD) immunoreactivities (Fig. 4). Figure 4 suggests that there is a proportional relationship between BCKD activity state and the ratio of pS293 to total E1{alpha} immunoreactivity.



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  		Fig. 4. Comparison of hepatic BCKD activity state and pS293 phosphorylation in PEG precipitates from livers of animals fed diets containing different protein concentrations. PEG precipitates were prepared from livers of animals fed diets containing 1 of 3 different protein concentrations as in Fig. 3. These samples were used to measure BCKD activities reported in Table 1 as well as pS293-specific and total E1{alpha} immunoreactivity by Western blotting. Graph shows the relationship between the percentage of the BCKD complex in the active state vs. the ratio of pS293 to total E1{alpha} immunoreactivity in liver samples. Western blots were analyzed by densitometry; however, similar results were obtained when the samples were later analyzed using the GeneGnome system.

 

We next evaluated the new pS293 antibody in a cell line, TREMK4 cells, expressing a recombinant form of BCKD kinase under the control of the tet-on promoter. In the presence of the tetracycline analog doxycycline, TREMK4 cells expressed large amounts of BCKD kinase, and this resulted in increased immunoreactivity of the pS293 antibody (Fig. 5, lane 3). Addition of 4x amino acids resulted in lower E1{alpha} phosphorylation and reduced pS293 immunoreactivity in control and doxycycline-treated cells (Fig. 5). Thus, even with high concentrations of BCKD kinase, amino acids significantly decreased site 1 phosphorylation. Overexpression of BCKD kinase did not affect the concentration of the E1{alpha} subunit (Fig. 5). Thus the antibody can assess the pS293 phosphorylation state in cell culture systems where enzymatic assay of BCKD activity is limited by the amount of cell protein.



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  		Fig. 5. Phospho-BCKD, total BCKD, and BCKD kinase in TREMK4 cells. TREMK4 cells were maintained as described previously in the absence (–) or presence (+) of doxycycline for 48 h. Cells were then incubated for 1 h with Krebs-Ringer-HEPES buffer (KRH) at 37°C before addition of fresh KRH containing (+) or lacking (–) 4x amino acids as indicated. Cell proteins were extracted with detergent containing lysate buffer and separated by SDS-PAGE, and antigens were detected by Western blotting (see EXPERIMENTAL PROCEDURES). Blot was probed with pS293 antibodies (top), followed by E1{alpha} antibodies (middle) and BCKD kinase antibodies (bottom). BCKD kinase antiserum reacted nonspecifically with a 2nd smaller protein visualized in TREMK4 cells.

 

Changes in BCKD phosphorylation after oral leucine administration. Frick et al. have shown that leucine regulates BCKD-mediated 14CO2 release from [1-14C]KIC, a measure of BCKD activity in adipocytes. In the absence of amino acids, BCKD activity in their study was very low (2.89 mU/g cell protein), and activity was shown to be associated with a high degree of BCKD phosphorylation as measured using 32P incorporation (15–17). Addition of 2.5 mM leucine increased adipocyte BCKD activity to 26 mU/g cell protein, and changes in activity correlated with decreased phosphorylation (17). On the basis of these observations, Frick et al. hypothesized that changes in circulating leucine might influence adipose tissue BCKD activity in vivo. To evaluate this hypothesis, we examined adipose tissue from food-deprived rats orally administered physiological saline or a leucine solution as described previously (45). This dose of leucine was sufficient to raise the plasma leucine concentrations to ~3 mM in 30 min. Figure 6 shows pS293 antibody immunoreactivity toward E1{alpha} from adipose tissue samples obtained from animals in the saline (S lanes) and leucine (L lanes) groups. Administration of leucine was associated with a dramatic decrease in pS293 immunoreactivity. This is the first demonstration of an effect of orally administered leucine on adipose tissue BCKD phosphorylation state.



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  		Fig. 6. Effect of orally administered leucine on anti-pS293 antibody immunoreactivity in adipose tissue. Adipose tissue was removed from rats 30 min after oral administration of PBS (S lanes) or leucine (L lanes) at 135 mg/100 g body wt, and tissue proteins were separated by SDS-PAGE and transferred to PVDF membranes as described in EXPERIMENTAL PROCEDURES. Blot was probed with purified anti-pS293 BCKD antibody. Each lane contained adipose tissue protein from a separate animal.

 

The results shown in Fig. 6 show that adipose tissue BCKD is highly phosphorylated, i.e., largely inactive, in vivo in food-deprived rats. To further evaluate this, we compared the pS293 immunoreactivity in adipose tissue to pS293 immunoreactivity in skeletal muscle (Fig. 7). BCKD is known to be mostly inactive in skeletal muscle (60). The gels were loaded with equivalent amounts of total BCKD E1{alpha} based on quantification using the GeneGnome ECL analysis system for Western blot quantification (not shown). The ratios of pS293 to E1{alpha} immunoreactivities in the phosphorylated BCKD standard, gastrocnemius, and adipose tissue extracts were not significantly different. Therefore, the results from Figs. 6 and 7 show that adipose tissue BCKD is inactive in fasting rats and that the enzyme can be activated after oral administration of leucine.



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  		Fig. 7. Anti-pS293 BCKD immunoreactivity in adipose tissue and gastrocnemius muscle from food-deprived rats. Top: representative Western blot of purified bovine dephosphorylated (D) and phosphorylated (P) BCKD complex as well as adipose tissue (A) and gastrocnemius (G) detergent-treated lysates. Equal amounts of total BCKD E1{alpha} subunit were loaded on the basis of concentrations obtained using Western blots quantified on the GeneGnome system (not shown). Proteins were separated by SDS-PAGE and transferred to PVDF membranes and probed with the pS293 antibody. Bottom: each bar represents mean ± SE of ratios of anti-pS293 BCKD antibody immunoreactivity to total E1{alpha} immunoreactivity from indicated tissues from 4 different animals.

 

Role of BCKD activity state in mTOR activation. The goal of the next experiment was to determine whether activation of leucine metabolism and mTOR signaling exhibited similar concentration dependencies. Plasma leucine concentrations and the phosphorylation states of BCKD (S293) and S6K1 (T389) in adipose tissue from different rats were measured 30 min after administration of saline or a dose of leucine. Figure 8 shows the plasma leucine concentrations after saline (0) and at each leucine dose (6.8–135 mg/100 g body wt). There was a linear relationship between plasma leucine concentration and leucine dose. The concentrations of most other amino acids, except BCAA, did not change appreciably in response to increasing leucine (Table 2). However, there was a trend toward a dose-dependent decrease in phenylalanine and tyrosine. This trend is consistent with an inhibitory effect of leucine on protein breakdown (8, 9), which increases with food deprivation.



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  		Fig. 8. Effect of oral leucine dose on plasma leucine concentrations. Different doses of leucine were orally administered, and truncal blood was collected after 30 min. Amino acid concentrations were measured by HPLC.

 

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  		Table 2. Effect of different doses of leucine on amino acid concentrations

 

Figure 9 shows that, at the lowest plasma leucine concentrations (i.e., 179, 267, and 548 µM), the ratio of pS293 to total E1{alpha} immunoreactivity was not significantly different. Phosphorylation at S293 declined gradually with increasing leucine dose, with a half-maximal dose of ~1,000 µM. On the other hand, T389 phosphorylation was more sensitive to leucine concentration and increased to a maximal level at a leucine dose that resulted in an average plasma leucine concentration of 548 µM. These findings indicate that dephosphorylation and activation of BCKD (leucine oxidation) are not required for activation of mTOR signaling.



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  		Fig. 9. Dose-dependent effects of leucine on ribosomal protein S6 kinase-1 (S6K1) phosphorylation at Thr389 (T389) and BCKD phosphorylation at S293. Adipose tissues were removed from rats 30 min after a dose of leucine and freeze clamped under liquid nitrogen. Proteins from detergent extracts of the tissue were separated on SDS-PAGE gels and transferred to PVDF membranes. A: results from blots probed with pT389 and total S6K antibodies. B: results from blots probed for pS293 and total BCKD E1{alpha} subunit. Immunoreactivity levels were quantified using the GeneGnome system. Means ± SD from >=3 animals at each dose are presented.

 


   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
DISCLOSURES
 REFERENCES
 
This study describes the first nonenzymatic measurement of the phosphorylation status of the BCKD enzyme complex and use of this new technique to test the hypothesis that leucine metabolism is required for leucine signaling in adipose tissue. Unlike the enzymatic assay, the pS293 antibody is sensitive enough to be used with samples that have low BCKD activity or where protein is limiting (10–20 µg of cell or tissue protein) and does not require PEG precipitation to concentrate the sample. The site 1 rat phosphorylation site (S293) on the E1{alpha} subunit that controls BCKD activity is highly conserved in mammalian species (see Fig. 1). Our results show that this antibody recognizes phosphorylated site 1 in rats, a hamster cell line, and the bovine enzyme. Therefore, the new pS293 antibody should be applicable to a wide variety of species.

Three different approaches were used to validate the pS293 antibody. First, we examined the immunoreactivity of phosphorylated and dephosphorylated purified BCKD complex from bovine kidney. Next, we examined pS293 immunoreactivity in PEG precipitates obtained from livers of rats maintained on diets with different protein contents, and we were able to show an excellent correlation between activity state and the ratio of pS293 to E1{alpha} immunoreactivity in samples of varying BCKD protein content (complex tissue changes). The ratios were compared with the activity state of the complex as measured by the enzymatic assay.

Liver was chosen to validate the pS293 antibody because of the well-documented relationship between BCKD activity state and dietary protein content (5, 19, 47). The pS293 antibody provided new insight into the mechanisms underlying dietary regulation of hepatic BCKD activity. Liver is thought to be the major site of BCAA oxidation, whereas BCAA transamination occurs in extrahepatic tissues. In rats fed a standard laboratory diet (>20% protein), which is more than sufficient to meet the animal's protein requirements, the liver enzyme is essentially active, with reported values for activity state of 82% (20% casein diet, Ref. 47), 94% (27% protein, Ref. 19), and 89% (after meal feeding 25% casein, Ref. 5). Only low-protein diets had a significant impact on activity state. On the other hand, increasing the protein content to 50% or higher did not always result in further increases in total BCKD activity above the standard diet. The results in Figs. 3 and 4 and Table 1 illustrate the complexity of the relationship between diet and activity. Dietary protein restriction results in increased expression of BCKD kinase (53), and high levels of kinase were found with BCKD in the PEG pellet, leading to increased steady-state levels of phosphorylated E1{alpha} and conservation of essential BCAA. Once dietary protein exceeded the requirement, little kinase was found with BCKD in the PEG pellet. Popov et al. (53) observed that deceasing dietary protein (50, 24, 0%) decreased BCKD kinase mRNA and activity (24, 25, 67). We also observed lower concentrations of E2 and E1 in the low-protein diet in addition to the changes in BCKD kinase. Despite the complexity of the dietary regulation of the BCKD complex, assessment of the ratio of pS293 and total E1{alpha} subunit immunoreactivities closely correlated with changes in BCKD activity based on actual activity measurements.

Finally, we examined the changes in pS293 immunoreactivity after drug-induced induction of BCKD kinase in TREMK-4 cells, where the activity state cannot be measured using the standard enzymatic assay even after PEG precipitation and use of radioactive KIC to increase assay sensitivity (data not shown). A new finding is that amino acid addition was highly effective at inhibiting BCKD phosphorylation regardless of drug induction of BCKD kinase, suggesting that excess BCKD kinase does not prevent activation of BCKD by keto acids arising from amino acid metabolism.

BCKD and S6K1 phosphorylation in adipose tissue. In the food-deprived state, adipose tissue BCKD S293 phosphorylation status was similar to that of gastrocnemius muscle, that is, mostly in the highly phosphorylated (i.e., inactive) form. This in vivo assessment agrees with previous in vitro activity measurements (17) indicating that <10% of the epididymal adipose tissue BCKD was active (~6% in some experiments). Thus the percentage of active BCKD in adipose tissue is similar to what has been reported for gastrocnemius. These findings imply that, although adipose tissue may have the capacity to oxidize the keto acid of leucine, this capacity is almost completely prevented by pS293 phosphorylation, as it is in muscle. This is in contrast to the situation in liver, in which most of the BCKD is active (dephosphorylated) in animals fed a standard protein-containing rat chow (excess protein), even after overnight food deprivation (19).

On the basis of their in vitro studies with leucine, Frick et al. (17) speculated that adipose tissue BCKD might be physiologically regulated in vivo by changes in circulating leucine concentrations after a meal. Our studies support this hypothesis, because we observed that, in animals fed sufficient leucine to raise the plasma leucine concentrations to ~3 mM, the enzyme was almost completely dephosphorylated, reflective of a highly active state. Again, this is similar to the in vitro results of Frick et al. When they incubated adipose tissue with 2.5 mM leucine, an 18-fold increase in BCKD activity was observed within 30 min. Thus our studies show that adipose tissue BCKD activity state is regulated by changes in leucine consumption. Therefore, BCKD kinase could potentially be a sensor for leucine after its rapid and reversible transamination to KIC via BCATm, in agreement with the attractive proposal of Xu et al. (65). However, when we compared the dose-dependent activation of BCKD and S6K1 in adipose tissue, we found that higher doses of leucine were required to produce a significant decrease in BCKD phosphorylation than was required to stimulate S6K1 phosphorylation at T389. This finding seems to be more consistent with a role for BCKD activation of the regulation of leucine/KIC metabolism than the initiation of mTOR signaling.

In conclusion, our findings continue to provide support for a role of leucine as a direct-acting nutrient signal as opposed to leucine metabolites. Leucine is well suited as a nutrient signal for several reasons. First, it is the most potent of the BCAA with regard to regulation of protein metabolism. Next, it is the most abundant essential amino acid in dietary protein, and mammals cannot synthesize it. A third special feature is that leucine cannot be metabolized directly by the liver, in contrast to its ability to actively metabolize other amino acids. This is a consequence of the lack of BCATm. The absence of BCATm from the liver may prevent "first-pass" losses of leucine and may thereby facilitate postprandial rises in plasma leucine concentration that would allow leucine to operate as a nutrient signal. The inability of liver to metabolize leucine is relevant to the hypothesis tested in this communication, because it has been demonstrated by several groups that leucine can regulate mTOR signaling in freshly isolated hepatocytes (6, 36, 37). It is unclear how leucine signaling to mTOR could be accomplished in hepatocytes if leucine metabolism was required to activate mTOR. Although further studies are required to rule out a role for leucine metabolism/oxidation in mTOR activation, our own findings, coupled with observations from the liver, seem to provide support for leucine rather than KIC, BCKD kinase, or leucine metabolism regulating mTOR.


   DISCLOSURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 DISCLOSURES
REFERENCES
 
This work was supported by grants from the National Institutes of Health (DK-62880, DK-53843, C. J. Lynch; GM-39277, T. C. Vary; DK-34738, S. M. Hutson), the US Department of Agriculture (98-35200-6067, S. M. Hutson), Solvay Pharmaceuticals, Germany (C. J. Lynch), and a research contract from GloboZymes, Inc. (Z. Damuni).


   FOOTNOTES
 

Address for reprint requests and other correspondence: C. J. Lynch, Dept. of Cell. & Mol. Physiology (MC H166, Rm C4757), Penn State College of Medicine, 500 University Dr., Hershey, PA 17033 (E-mail: clynch{at}psu.edu).

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.

Present address of Z. Damuni: GloboZymes, 6351 Corte del Abeto, Ste. 101-A, Carlsbad, CA 92009.


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 DISCUSSION
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