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Am J Physiol Endocrinol Metab 293: E1552-E1563, 2007. First published October 9, 2007; doi:10.1152/ajpendo.00134.2007
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Obesity-related elevations in plasma leucine are associated with alterations in enzymes involved in branched-chain amino acid metabolism

Pengxiang She,1 Cynthia Van Horn,3 Tanya Reid,3 Susan M. Hutson,3 Robert N. Cooney,1,2 and Christopher J. Lynch1

Departments of 1Cellular and Molecular Physiology and 2Surgery, College of Medicine, Pennsylvania State University, Hershey, Pennsylvania; and 3Department of Biochemistry and Molecular Biology, Nutrition Research Center, Wake Forest University Health Sciences, Winston-Salem, North Carolina

Submitted 27 February 2007 ; accepted in final form 4 October 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 DISCLAIMER
 REFERENCES
 
Elevations in branched-chain amino acids (BCAAs) in human obesity were first reported in the 1960s. Such reports are of interest because of the emerging role of BCAAs as potential regulators of satiety, leptin, glucose, cell signaling, adiposity, and body weight (mTOR and PKC). To explore loss of catabolic capacity as a potential contributor to the obesity-related rises in BCAAs, we assessed the first two enzymatic steps, catalyzed by mitochondrial branched chain amino acid aminotransferase (BCATm) or the branched chain {alpha}-keto acid dehydrogenase (BCKD E1{alpha} subunit) complex, in two rodent models of obesity (ob/ob mice and Zucker rats) and after surgical weight loss intervention in humans. Obese rodents exhibited hyperaminoacidemia including BCAAs. Whereas no obesity-related changes were observed in rodent skeletal muscle BCATm, pS293, or total BCKD E1{alpha} or BCKD kinase, in liver BCKD E1{alpha} was either unaltered or diminished by obesity, and pS293 (associated with the inactive state of BCKD) increased, along with BCKD kinase. In epididymal fat, obesity-related declines were observed in BCATm and BCKD E1{alpha}. Plasma BCAAs were diminished by an overnight fast coinciding with dissipation of the changes in adipose tissue but not in liver. BCAAs also were reduced by surgical weight loss intervention (Roux-en-Y gastric bypass) in human subjects studied longitudinally. These changes coincided with increased BCATm and BCKD E1{alpha} in omental and subcutaneous fat. Our results are consistent with the idea that tissue-specific alterations in BCAA metabolism, in liver and adipose tissue but not in muscle, may contribute to the rise in plasma BCAAs in obesity.

obesity; mitochondrial branched chain amino acid transaminase; branched chain keto acid dehydrogenase; branched chain keto acid dehydrogenase kinase; ob/ob mice; Zucker rats; bariatric surgery; humans

PLASMA CONCENTRATIONS of branched-chain amino acids (BCAAs) are elevated in humans and animal models of obesity (4, 11, 12, 31, 37, 46, 48, 52, 56). Although the plasma levels of other amino acids may also change in obesity, the rises in the BCAAs are of particular interest because they appear to have unique obesity-related effects. Indeed, it has been posited that BCAAs may be responsible for some of the beneficial effects of high-protein diets, improving body weight control and adiposity (1, 9, 18, 29, 30, 39). Similarly, BCAAs improve muscle glucose uptake, whole body glucose metabolism, and oxidation (7, 8, 43). Furthermore, they have been shown to regulate leptin secretion from fat and food intake at the level of the hypothalamus via mTOR (mammalian target of rapamycin) signaling, which also has been implicated in obesity (6, 13, 28, 32, 34, 47). Finally, rises in plasma BCAAs due to a block in mitochondrial branched chain amino acid aminotransferase (BCATm) have also been associated with improvements in glucose tolerance and resistance to diet-induced obesity (50). Consequently, an improved understanding of the mechanism(s) underlying obesity-related rises in BCAAs is important. BCAAs might be elevated simply because obese individuals eat more food; however, in humans, rises in BCAAs are still seen after an overnight fast (11, 12). Another potential explanation for the rise in BCAAs in obesity is increased protein catabolism secondary to insulin resistance. Jensen and Haymond (26) reported that proteolysis is elevated in moderate upper body obesity and that insulin's antiproteolytic action is impaired. Luzi et al. (33) also concluded that the increased proteolysis in obesity was a result of impaired antiproteolytic action of insulin. An idea that has received less attention is that a reduction in BCAA metabolism might be involved (31). In lean animals, BCAA metabolism is highly regulated by changes in cellular BCAA concentrations due to alterations in dietary intake (20, 51). Studies in transgenic mice with disruption of BCATm or branched chain {alpha}-keto acid dehydrogenase kinase (BCKD kinase) support the idea that dysregulation of BCAA metabolism results in sustained changes in plasma BCAA concentrations (27, 50). Furthermore, in other nutritional states in which BCAAs are elevated, such as starvation or protein malnutrition, BCAA metabolism and proteolysis both may contribute to changes in BCAAs (20, 51). Given the robust regulation and capacity of the BCAA metabolic pathway, it seems unlikely that a rise in the plasma concentration of BCAAs (e.g., due to increased proteolysis or food intake) in obesity could be sustained in the absence of some alteration in BCAA disposal. In support of this idea, microarray studies have suggested that mRNA for enzymes involved in BCAA metabolism in adipose tissue are depressed in mutant or transgenic animals with an obese phenotype (21, 40).

The current study examines the possibility that key enzyme components of BCAA metabolism are altered in obesity. We examined the first two steps in the catabolic pathway catalyzed by the BCATm and BCKD enzyme complex. BCATm catalyzes the first step of BCAA metabolism in the mitochondrial matrix in most tissues outside the central nervous system (54). It transfers the amino group of a BCAA to {alpha}-ketoglutarate to form glutamate and the corresponding branched chain {alpha}-keto acid, for example, {alpha}-ketoisocaproate (KIC), from leucine. This reaction is rapid and of high capacity in muscle and fat. The second step is regulated by the BCKD complex. The complex catalyzes oxidative decarboxylation of the branched chain {alpha}-keto acids, forming the branched-chain acyl-CoA derivatives, CO2, and NADH. The multicomponent enzyme complex contains three enzymes, an associated kinase, and phosphatase (19). E1 catalyzes oxidative decarboxylation of the branched chain {alpha}-keto acids and has an {alpha}2β2 structure with a covalently bound thiamine pyrophosphate cofactor. E2 is the dihydrolipoyl transacylase, and E3 is a flavin-linked dihydrolipoyl dehydrogenase that is not unique to the BCKD complex (19). Because BCATm is not expressed in rodent liver, BCAAs from dietary protein bypass first metabolism in liver. This may contribute to the sharp rise of plasma leucine in response to a meal, thereby promoting leucine signaling in peripheral tissues that respond to leucine (34). BCKD kinase phosphorylates the E1{alpha} subunit of BCKD at two sites, termed sites 1 and 2 (45). Site-directed mutagenesis studies (60) have shown that phosphorylation at site 1 [rat E1{alpha}-Ser293 (S293{alpha})] inhibits BCKD activity. The importance of BCKD kinase in the regulation of BCKD was recently demonstrated in BCKD kinase knockout mice in which BCKD activity and BCAA catabolism were elevated in all tissues except liver, and BCAA concentrations in plasma and various tissues were markedly decreased (27). Changes in the expression of the hepatic BCKD kinase occur in metabolic states associated with changes in dietary protein intake or plasma thyroid hormone concentrations (19).

To examine the potential role of BCAA metabolism in obesity-associated elevations in plasma BCAAs, we examined BCATm and/or BCKD complex concentrations and BCKD E1{alpha} phosphorylation status in muscle, liver, and fat from obese rodents. We also examined these enzymes in visceral and subcutaneous adipose tissue from morbidly obese humans before and after a weight loss intervention, gastric bypass surgery. The results are consistent with the hypothesis that alterations in BCAA metabolism in liver and adipose tissue may contribute to the rise in BCAAs in obesity.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 DISCLAIMER
 REFERENCES
 
Animals and human subjects. All animal experiments were reviewed and approved by the Animal Care and Use Committee at the Pennsylvania State University College of Medicine and adhered to the National Institutes of Health guidelines for the care and use of experimental animals. Mice (ob/ob, lean controls, and C57BL/6J) were purchased from Jackson Laboratory. Zucker fatty and lean control rats were purchased from Charles River Laboratory. Animals were housed in the Pennsylvania State College of Medicine animal facility with an ambient temperature of 21–23°C on a 12:12-h light-dark cycle and given free access to water and a rodent chow diet (Harlan Teklad 2018; Madison, Wisconsin). Tissues were obtained from freely fed or overnight-fasted animals that were euthanized with pentobarbital (100–150 mg/kg) and clamped between plates cooled to the temperature of liquid nitrogen.

Anonymous human tissue samples were obtained from the Pennsylvania Department of Health obesity tissue bank. Recruitment of the subjects and operation of the tissue bank is approved by the College of Medicine Institutional Review Board. Plasma and adipose tissue (subcutaneous and omental) samples were obtained for male and female subjects who underwent open gastric bypass surgery for treatment of morbid obesity. Informed consent was obtained from these subjects before surgery. Subjects were ingested nothing by mouth after midnight the day before surgery and tissue collection. Two samples were collected from each subject, one at the initial bariatric surgery and the other, in a smaller number of subjects, at a second surgical procedure performed an average of 17 mo after the first surgery. The second surgery was medically necessary for hernia repair, gall bladder disease, or excess skin removal.

BCAT and BCKD enzyme activity assays. Frozen powdered adipose tissue was suspended in buffer (1 g per 3 ml of extraction buffer) containing 25 mM HEPES (pH 7.4), 0.4% 3-([3-cholamidopropyl]dimethylammonio)-1-propanesulfonate (CHAPS), 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol (DTT), and protease inhibitors (53). The tissues suspension was subjected to freeze-thaw sonication and centrifuged at 10,000 gfor 30 min. The supernatant was assayed for BCAT activity as described previously (23). Briefly, activity was measured at 37°C in 25 mM potassium phosphate buffer, pH 7.8, which contained 50 µM pyridoxal phosphate, 5 mM DTT, 12 mM isoleucine, and 1 mM {alpha}-keto-[1-14C]isovalerate (KIV). A unit of activity was defined as 1 µmol of valine formed per minute at 37°C. Tissue extraction and determination of BCKD activity were carried out as described previously (41). BCKD complex was precipitated from whole tissue extracts using 9% polyethylene glycol. BCKD activity was determined spectrophotometrically by measuring the rate of NADH production resulting from the conversion of KIV to isobutyryl-CoA. A unit of enzyme activity was defined as 1 µmol of NADH formed per minute at 30°C. BCKD activity was confirmed by the radioactive assay, which measured 14CO2 release from [1-14C]KIV. A unit of activity was defined as 1 µmol of 14CO2 formed per minute at 30°C.

Western blot analysis. Equivalent weights of frozen powdered liver, muscle, or adipose tissues were homogenized on ice using a Polytron in either 3 (adipose tissue) or 7 volumes of a phospho-preserving homogenization buffer. The components of that buffer were (in mM) 20 HEPES, pH 7.4 (buffer pH adjusted with 1 or 10 mM NaOH), 2 EGTA, 50 NaF, 100 KCl, 0.2 EDTA, 50 β-glycerophosphate, 0.1 4-(2-aminoethyl)benzenesulfonyl fluoride-HCl, 1 benzamidine, 0.5 sodium vanadate, 1 M microcystin LR, 0.4% CHAPS, and 1% Triton X-100. The homogenate was centrifuged at 10,000 g for 10 min at 4°C. An aliquot of the supernatant was reserved for protein assay using the BCA protein assay kit (Pierce, Rockford, IL), and the rest was added to an equal volume of 2x or 5x Laemmli sodium dodecyl sulfate (SDS) sample buffer. After the protein concentration in the homogenates was assayed, equal amounts of protein were then electrophoresed on Tris·HCl gels and proteins were transferred to polyvinylidene difluoride membranes. Western blots of the transferred proteins were conducted as previously described (35, 54). Densitometric analysis of immunoblots was performed using NIH ImageJ. The antibodies against BCATm (41.7 kDA), total BCKD E1 (45.5 kDA), and phospho-[C-YRIGHH-(phospho-Ser)-TSDDSS] peptide corresponding to phosphorylation site 1 on the E1{alpha} subunit of BCKD E1 (45.5 kDa) were generated and characterized as previously described (35, 54). To determine the ratio of phospho-S293 (pS293) to E1{alpha} when the amount of E1{alpha} was found to be significantly different between conditions, the samples were re-electrophoresed and analyzed by loading approximately the same amount of total BCKD E1{alpha} on the SDS-PAGE gels. Resulting blots were immunoblotted for pS293 immunoreactivity, followed by stripping and reprobing for total E1{alpha}. BCKD kinase (46.1 kDA) was monitored with previously described polyclonal antibodies (53) and with monoclonal antibodies, the latter being a generous gift from Dr. Yoshiharu Shimomura (Nagoya Institute of Technology, Nagoya, Japan). Loading control antibodies were to β-actin (42 kDa; Sigma Aldrich, St. Louis, MO) and tubulin (55 kDa; Thermoscientific, Freemont, CA).

Analytical procedures. Plasma BCAA concentrations were measured using either an enzymatic spectrophotometric assay as described by Beckett (2) or as part of a complete amino acid determination using an HPLC-coupled fluorometric method (57). For the spectrophotometric assay, bacterial leucine dehydrogenase (Toyobo, New York, NY) was used to catalyze the oxidation of BCAAs. Production of NADH was measured at 340 nm using a spectrophotometer.

For the HPLC assay of amino acids, the method described by Wu and Knabe (57) was used. Plasma was deproteinized with an equal volume of 1.5 M HClO4, and the resulting supernatant was neutralized with 2 M K2CO3. Before amino acid analysis, 0.1 ml of the neutralized plasma extract was mixed with 0.1 ml of a 1.2% benzoic acid solution (prepared with saturated K2B4O7) and brought to a total volume of 1.6 ml with HPLC-grade H2O. Standards were subjected to the same protocol with final concentrations ranging from 0.01 to 1.875 nmol/ml. The sample solution (25 µl) was mixed with 25 µl of o-phthaldialdehyde reagent (50 mg of o-phthaldialdehyde in 1.25 ml of methanol to which 11.2 ml of 0.04 M sodium borate buffer, pH 9.5, 50 µl of mercaptoethanol, and 0.4 ml of Brij-35 were added). After mixing, the solution was incubated for 2 min in the WATERS 2695 HPLC system before injection and separation of the o-phthaldialdehyde derivatives. Separation was made by gradient elution from a Supelcosil LC-18 column (15 cm x 4.6 mm, 3 µM; Sigma) with a mobile phase of 86% solvent A (0.1 M sodium acetate, pH 7.2, containing 9% methanol and 0.5% tetrahydrofuran) and 14% of solvent B (100% methanol). The gradient used excitation and emission wavelengths that are described in Ref. 57. Detection was made with a JASCO FP-152 detector.

Blood glucose was measured using an OneTouch Ultra glucometer (Lifescan/Johnson & Johnson, Milpitas, CA). Plasma insulin concentrations were measured using human, rat, or mouse insulin-specific ELISA kits (ALPCO Diagnostics, Salem, NH) as appropriate for the species. Plasma urea was measured using the Vitros chemistry system (DT60 II and DTSc II modules; Ortho-Clinical Diagnostics, Rochester, NY).

Statistical analysis. Initial studies compared nutritional states separately; that is, fed and fasted data were initially derived from two separate blots at different times. However, some differences in the regulation of endpoints between the fed and fasted states made it necessary to reanalyze stored samples together on the same blots, where possible. To compare them together, the remaining samples were electrophoresed on the same blot and reanalyzed together; however, there was insufficient material to rerun all of the ob/ob samples. In that case, the original data are shown for those endpoints, and a two-tailed Student's t-test was used to compare the lean and obese states only (P < 0.05 was taken as showing statistical significance). The other reanalyzed samples were compared between lean and obese as well as nutritional status using an ANOVA, followed automatically, when statistical differences were observed, with a Student-Newman-Keuls multiple comparisons post test (values are means ± SE, and P < 0.05 was considered significantly different). The statistical analyses were conducted using Prism and Instat computer programs (GraphPad Software, San Diego, CA).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 DISCLAIMER
 REFERENCES
 
Plasma amino acids. At 13–14 wk of age, ob/ob mice exhibited severe obesity compared with their lean counterparts. This was reflected in their body, epididymal fat pad, and liver weights, which were 1.7-, 8-, and 2.5-fold greater, respectively, than in lean animals (Table 1). In addition, their gastrocnemius muscle and heart weights were significantly diminished. As shown by the ~10-fold increase in fasting plasma insulin concentrations, the obese ob/ob mice also had severe insulin resistance (Table 1). Plasma BCAAs measured using either the spectrophotometric or HPLC methods were elevated in the postabsorptive state; however, the differences tended to decline with food deprivation in ob/ob mice and Zucker rats (Fig. 1 A and Table 2). Thus, compared with control lean animals, combined plasma BCAA concentrations were 89 and 26% higher in the postabsorptive (blood collected at ~10:00 AM in random-fed animals) and overnight-fasted animals, respectively (Fig. 1A). In agreement with a previous report (46), total BCAA concentrations were 48% higher in fed obese Zucker rats compared with Zucker lean rats (Fig. 1B). Plasma BCAA concentrations were greatly depressed by overnight food deprivation and not statistically different. In contrast to the elevated BCAAs in obesity, fed plasma glucose concentrations were not significantly different between lean and obese animals (Table 1). The results in the fasted ob/ob mice and previous studies on fasted humans suggest that the obesity-related differences in plasma BCAAs are not due solely to higher food intake in obese animals.


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  		Table 1. Body and organ weights and plasma concentration of insulin and metabolites in lean and ob/ob mice

 

Figure 1
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  		Fig. 1. Effect of obesity on plasma branched-chain amino acid (BCAA) concentrations. Plasma BCAAs were measured using spectrophotometric assay. A: randomly fed (fed) and overnight food-deprived (fasted) plasma BCAA concentrations in 13-wk-old ob/ob male and age-matched lean control mice fed a normal chow diet (n = 8/group). *P < 0.05; ***P < 0.001 vs. control in the same nutritional state. B: fed and fasted plasma BCAA concentrations in ~13-wk-old Zucker fatty and age-matched lean control rats (n = 10/group). **P < 0.01 vs. lean controls in the same nutritional state.

 

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  		Table 2. Plasma amino acid concentrations in lean and ob/ob mice

 
As shown in Table 2, in addition to the BCAAs, obesity had effects on other amino acids. In the postabsorptive state, the largest changes were observed in the large neutral amino acids, which include the aromatic amino acids (Phe, Tyr, and Trp) and BCAAs (Leu, Ile, and Val) with concentrations ranging from 56 to 84% higher in ob/ob mice compared with the lean control values. The urea cycle intermediates ornithine (Orn) and citrulline (Cit) were elevated by 96 and 39%, respectively, whereas plasma Arg concentrations were lower (–24%) than in lean controls. Plasma levels of dispensable amino acids Ser, Asn, and Thr also were significantly higher (~30%) in fed obese animals than in lean controls. In the fasting state, plasma concentrations of seven amino acids were less, and four amino acids were greater, in ob/ob mice than in control mice. Notably, plasma concentrations of Orn, Trp, and Ile were elevated in both fed and fasting states in ob/ob mice compared with lean mice. The gluconeogenic amino acids Ser, Thr, and Ala, which were elevated in the fed state, were lower in obese ob/ob animals than in lean controls during fasting. Gly was also lower than in lean controls during fasting.

Obesity-related differences in the plasma concentrations of amino acids were sometimes dependent on whether the plasma was from random-fed or overnight food-deprived animals (Table 2). In lean controls, concentrations of most plasma amino acids did not change significantly in response to an overnight fast. Exceptions were Glu, Asn, taurine, Tyr, Phe, and the urea cycle intermediate Orn. Food deprivation had a greater effect on the plasma amino acid pattern in the obese ob/ob mice, with most amino acids showing statistically significant decreases with respect to fed animals. Plasma BCAA changes in response to fasting were different in lean controls and ob/ob mice (Fig. 1A and Table 1). Compared with the fed levels, fasting plasma BCAAs did not differ in lean animals; however, they were decreased by 30% (P < 0.001) in the ob/ob mice. Thus the BCAAs in obese animals were more sensitive to fasting than those in the lean controls.

Table 3 shows the plasma concentrations of the transamination products of BCAAs, the branched-chain {alpha}-keto acids, in the fed and fasted state in ob/ob mice and lean controls. In the fed state, plasma branched-chain {alpha}-keto acids were higher in ob/ob mice compared with lean animals with the increases in the {alpha}-keto acid of leucine (KIC) reaching statistical significance. Consistent with a previous report in rats (24), in lean mice plasma branched-chain {alpha}-keto acids were elevated by fasting compared with the fed state (P < 0.001). In ob/ob mice, fasting plasma branched-chain {alpha}-keto acids also were elevated compared with fed obese mice (P < 0.006 and P < 0.05 for KIV and KIC, respectively).


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  		Table 3. Plasma concentrations of branched-chain {alpha}-keto acids

 
The obesity-related differences in plasma BCAAs may not be due solely to higher food intake in obese animals. For example, whereas the obesity-related changes in BCAAs became statistically insignificant with fasting in Zucker rats, in ob/ob mice plasma BCAAs were still elevated in the fasted state (Fig. 1 and Table 2), and two of the keto acids were also elevated in fasted ob/ob mice. As mentioned earlier, BCAAs are also elevated in other obesity models as well as fasted obese humans (11, 12, 31). To begin to understand the molecular basis for the elevated plasma BCAA concentrations in obese animals, we examined the protein concentrations and activities of the enzymes catalyzing the first two key steps in body BCAA metabolism: BCATm and the BCKD enzyme complex in lean and obese ob/ob mice and Zucker rats.

Effect of obesity on enzymes in the BCAA catabolic pathway in skeletal muscle, liver, and adipose tissue. Because of its large mass (~40% of body weight in lean animals) and high level of BCATm activity relative to BCKD activity, skeletal muscle plays a significant role in body BCAA transamination and is a major source of plasma branched-chain {alpha}-keto acids (24). However, when ob/ob mice and their sibling lean controls were compared, no differences were observed in the relative concentrations of skeletal muscle BCATm, BCKD E1{alpha} subunit, BCKD kinase, or E1{alpha}-pS293 phosphorylation (Fig. 2). These results suggest that BCAA metabolism in skeletal muscle is unaltered in the obese state and there is limited oxidation of BCAAs in muscle as observed in nonobese animals, and it is likely that skeletal muscle is a major source of plasma BCKAs in obese animals as well as lean animals. Similar results were found in the skeletal muscle of Zucker fatty rats (Fig. 2).


Figure 2
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  		Fig. 2. BCAA catabolic enzymes and phospho-Ser293 (pS293) E1{alpha} immunoreactivity in skeletal muscle of ob/ob mice and Zucker rats. Gastrocnemius muscle was obtained from random-fed or overnight-fasted ob/ob mice and Zucker rats. Equal amounts of muscle lysate protein from lean (L) control and obese (O) mice (A) or lean (L) and obese "fatty" (F) Zucker rats (B) were then separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF). These blots were probed for antibodies against mitochondrial branched chain amino acid aminotransferase (BCATm), branched chain {alpha}-keto acid dehydrogenase (BCKD E1{alpha} subunit), phosphorylated BCKD (pS293 E1{alpha}), and BCKD kinase (bottom band is nonspecific). Tubulin was used as a loading control. No statistical difference was found in the concentration of any of the antigens between the lean and obese state when replicates were analyzed (P > 0.05, n = 8; densitometry not shown).

 
BCATm is not expressed in adult rodent liver (23, 25), whereas liver contains the highest concentration of BCKD complex activity and is thought to be the major site of branched-chain {alpha}-keto acid oxidation (53). On a standard rodent chow diet, liver BCKD complex is nearly 100% unphosphorylated in the active state and has low BCKD kinase activity, i.e., the E1{alpha} subunit Ser293 is largely unphosphorylated (17, 19, 35). As in muscle, amounts of BCKD E1{alpha} subunit were unaltered by obesity in ob/ob mice compared with lean controls (Fig. 3A). However, BCKD kinase expression was elevated two- to threefold in fed or overnight-fasted ob/ob mice compared with lean controls (Fig. 3D). The increase in kinase expression was associated with a two- to threefold increase in the ratio of E1{alpha}-pS293 phosphorylation to total E1{alpha} in both fed and overnight-fasted livers from ob/ob compared with lean sibling control mice (Fig. 3C). We have shown previously a strong correlation between decreases in liver BCKD activity and immunoreactivity of the pS293 antibody in rats; however, we have not conducted similar experiments in mice. Therefore, BCKD activity was examined. Hepatic BCKD activity was depressed in obese ob/ob mice based on either grams of tissue weight or milligrams of protein (Table 4).


Figure 3
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  		Fig. 3. Total and pS293 phosphorylated BCKD E1{alpha} and BCKD kinase immunoreactivity in liver from ob/ob mice. Liver protein lysates, made from frozen lean and ob/ob mouse liver powders, were equalized for protein content, separated by SDS-PAGE, and transferred to PVDF for immunoblotting. A: representative immunoblots of total BCKD E1{alpha} (liver does not express BCATm), pS293 E1{alpha}, BCKD kinase. Western blotting for β-actin was used as a loading control. Graphs show densitometric analysis of replicates for BCKD E1{alpha} (B), pS293 E1{alpha}/BCKD E1{alpha} ratio (C), and BCKD kinase (D). Fed and fasted data were initially derived from 2 separate blots at different times. To compare them together, the remaining samples were electrophoresed on the same blot and reanalyzed together; however, there was insufficient material to rerun the BCKD kinase. The original data are shown for that endpoint; a t-test was used to compare the lean and obese states only (n = 6/group). The other reanalyzed samples were compared between lean and obese as well as nutritional status using an ANOVA, followed by a Student-Newman-Keuls multiple comparisons post test. **P < 0.01; ***P < 0.001, lean vs. obese condition in the corresponding nutritional state. No differences were observed between the lean and fasted states for any antigen. The densitometry values were 7,539 ± 665, 7,358 ± 692, 6,835 ± 532, and 6,833 ± 578 for fed lean, fed obese, fasted lean, and fasted obese mouse liver, respectively (P > 0.05).

 

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  		Table 4. BCKD activity in livers from lean control and ob/ob mice in the fed and fasted state

 
Overnight fasting does not normally have an effect on the phosphorylation state and activity of BCKD in rats, whereas longer term starvation results in diminished BCKD activity (19). Indeed, there was a trend toward lower levels of total BCKD E1{alpha} subunit in overnight-fasted mice, but differences were not statistically significant. Nevertheless, average BCKD activity tended to be lower for both groups (Table 4). Thus an overnight fast in mice may be more representative of a starvation response in rats in terms of this activity. The lowered hepatic BCKD activity during fasting compared with the fed state is consistent with elevated branched-chain {alpha}-keto acids during fasting (Table 3) and the major role of liver in branched-chain {alpha}-keto acid oxidation in lean animals (53).

Changes in hepatic BCKD complex enzymes also were observed in Zucker rats. In contrast to ob/ob mouse liver, however, statistical changes occurred in both total BCKD and its phosphorylation state (Fig. 4). Total BCKD E1{alpha} protein level was decreased by over 30% in the fed and fasted state by obesity in Zucker fatty livers (GoFig. 6A). Despite the decrease in total BCKD E1{alpha} as observed in ob/ob animals, the ratio of pS293-E1{alpha} to E1{alpha} were also elevated approximately twofold (Fig. 4B). This was also independent of nutritional status. Consistent with changes in ob/ob mouse liver, hepatic BCKD kinase expression was elevated approximately twofold in both fed and overnight-fasted Zucker fatty rats compared with lean controls (Fig. 4C). This is a potential explanation for the increased S293-E1{alpha} phosphorylation we observed.


Figure 4
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  		Fig. 4. Total and pS293 phosphorylated BCKD E1{alpha} and BCKD kinase immunoreactivity in liver from Zucker rats. Liver lysates, made from frozen lean and fa/fa fatty Zucker rat liver powders, were equalized for protein content, separated by SDS-PAGE, and transferred to PVDF. A: representative immunoblots of BCKD E1{alpha} (liver does not express BCATm), pS293 E1{alpha}, BCKD kinase. Western blotting for β-actin was used as a loading control. Graphs show densitometric analysis of blots for BCKD E1{alpha} (B), pS293 E1{alpha}/BCKD E1{alpha} ratio (C), and BCKD kinase (D). An ANOVA was first used to compare the lean and obese states as well as the fed and fasted states, followed by a Student-Newman-Keuls multiple comparisons post test. *P < 0.05; **P < 0.01; ***P < 0.001, fatty vs. lean condition in the corresponding nutritional state. No statistical differences were detected between expression levels of any of the antigens when random fed and fasted states were compared. The densitometry values were 6,270 ± 703, 6,198 ± 255, 6,297 ± 20, and 6,786 ± 599 for fed lean, fed obese, fasted lean, and fasted obese rat liver, respectively (P > 0.05).

 

Figure 5
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  		Fig. 5. BCATm, total and pS293 phosphorylated BCKD E1{alpha}, and BCKD kinase immunoreactivity in adipose tissue from ob/ob mice. Equal amounts of protein lysates, made from frozen lean and obese ob/ob mice adipose tissue powders, were separated by SDS-PAGE and transferred to PVDF. A: representative immunoblots of BCATm, total BCKD E1{alpha}, pS293 E1{alpha}, and BCKD kinase. Western blotting for β-actin was used as a loading control. Graphs show densitometric analysis of BCATm (B), total BCKD E1{alpha} (C), pS293 E1{alpha} immunoreactivity as a ratio to total BCKD (D), and BCKD E1{alpha} kinase (E). An ANOVA followed by a Student-Newman-Keuls multiple comparisons post test was used to compare the lean and obese states across the fed and fasted states (n = 6/group). **P < 0.01; ***P < 0.001, obese vs. lean mice. cP < 0.05 vs. fed lean control. oP < 0.01 vs. fed obese condition. The densitometry values were 8,293 ± 493, 8,318 ± 517, 8,170 ± 337, and 7,712 ± 202 for fed lean, fed obese, fasted lean, and fasted obese mouse adipose, respectively (P > 0.05).

 

Figure 6
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  		Fig. 6. Expression of BCATm, pS293 phosphorylated and total BCKD E1{alpha}, and BCKD kinase immunoreactivity in adipose tissue of Zucker rats. Equal amounts of protein lysates, made from frozen lean and obese Zucker rat adipose tissue powders, were separated by SDS-PAGE and transferred to PVDF. A: representative immunoblots of BCATm, total BCKD E1{alpha}, pS293 E1{alpha}, and BCKD kinase. Western blotting for β-actin was used as a loading control. Graphs show densitometric analysis of BCATm (B), total BCKD E1{alpha} (C), pS293 E1{alpha} immunoreactivity as a ratio to total BCKD (D), and BCKD E1{alpha} kinase (E). An ANOVA followed by a Student-Newman-Keuls multiple comparisons post test was used to compare the lean and fatty states across the fed and fasted states (n = 6/group). *P < 0.05; **P < 0.01, obese vs. lean mice. fP < 0.05 vs. fed obese condition. The densitometry values were 5,876 ± 232, 6,122 ± 550, 8,331 ± 251, and 8,029 ± 347 for fed lean, fed obese, fasted lean, and fasted obese rat adipose, respectively (P > 0.05).

 
It is unclear what relative contribution adipose tissue makes to whole body BCAA metabolism and how this changes in obesity, where adipose tissue becomes a more significant contributor to body weight. Adipose tissue contains BCATm and BCKD activity (14, 16). As shown by Western blotting, adipose tissue has about twice as much BCATm per milligram of protein as muscle and about five times more BCKD E1{alpha} subunit (36). The amount of adipose tissue E1{alpha} subunit is comparable to that in liver (36). In Western blots, relative concentrations of BCATm protein were lower in various preparations of fed ob/ob mouse adipose tissue compared with lean controls (Fig. 5, A and B), with the smallest difference being around a 63% reduction. BCAT activity per gram of tissue was 52% lower in obese ob/ob mice (129 ± 6 nmol·min–1·g tissue–1, n = 8) than in lean controls (266 ± 26 nmol·min–1·g tissue–1, n = 6, P < 0.01). When adjusted for differences in protein content of the tissue, BCAT activity was still 30% lower in the ob/ob mice (27 ± 2 nmol·min–1·mg protein–1) than in the lean controls (19 ± 1 nmol·min–1·mg protein–1, n = 6, P < 0.05). Similarly, relative concentrations of adipose tissue BCATm protein were 3.8-fold lower in Zucker fatty rats compared with lean controls (Fig. 6, A and B). A surprising finding in ob/ob mice adipose tissue, also observed in the Zucker rats, was that the obesity-related changes in BCATm differences disappeared in response to fasting, with similar concentrations of BCATm found in obese and lean animals. Thus fasted ob/ob mice had higher BCATm per milligram of protein relative to fed obese mice, but not to the levels observed in lean fed control mice.

Adipose tissue BCKD E1{alpha} protein expression in random-fed ob/ob mice and Zucker fatty rats was 2.2- and 3.4-fold lower, respectively, than in lean controls (Figs. 5C and 6C). In contrast, the relative concentrations of BCKD kinase were approximately twofold higher in adipose tissue from ob/ob mice and Zucker fatty rats compared with lean animals (Figs. 5E and 6E). Because BCKD kinase is a mitochondrial protein, like BCATm and E1{alpha}, the obesity-related declines in BCKD and BCATm protein levels are not likely to be related to a result of a loss of mitochondria per se.

Despite increases in adipose tissue BCKD kinase, S293-BCKD E1{alpha} phosphorylation was less per milligram of protein in both the ob/ob mice and Zucker fatty rats (Figs. 5A and 6A), and changes in the ratio of pS293 to total E1{alpha} were not significant (Figs. 5D and 6D). This is likely a consequence of the decrease in total BCKD and the fact that normally adipose tissue is already extensively phosphorylated, in contrast to the liver (15, 35).

Effect of weight loss intervention. Banked human samples also were available to examine the effect of a weight loss intervention on plasma BCAAs and adipose tissue BCATm and BCKD E1{alpha} subunit (Figs. 7 and 8). Longitudinal samples were collected from overnight-fasted human volunteers with morbid obesity at the time of bariatric surgery (Roux-en-Y gastric bypass) and at a subsequent second surgery for panniculectomy, adhesions, or hernia repair. The samples were blood plasma, subcutaneous adipose tissue, and omental (a visceral depot) adipose tissue.


Figure 7
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  		Fig. 7. Plasma BCAA concentrations in overnight-fasted morbidly obese human subjects before and after gastric bypass surgery. Plasma was collected between 9:00 AM and 12:00 PM after an overnight fast following induction of general anesthesia from subjects described in Table 4 before Roux-en-Y gastric bypass surgery and later when subjects returned for a second surgery. Plasma was stored at –80°C until assay for total BCAA concentration was conducted as described in Fig. 1 using an enzymatic assay. **P < 0.01, before vs. after surgery.

 

Figure 8
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  		Fig. 8. Subcutaneous and visceral adipose tissue protein expression before and after bariatric surgery-induced weight loss. Abdominal subcutaneous and visceral (omental) adipose tissue was collected longitudinally from subjects described in Table 5. The freeze-clamped adipose tissue was powdered under liquid nitrogen. Infranatants of lysates prepared from these under phosphorylation state-preserving conditions were solubilized with SDS-PAGE sample buffer, separated by SDS gel electrophoresis, and transferred to PVDF. A: representative immunoblots of BCATm, total BCKD E1{alpha}, pS293 E1{alpha}, and BCKD kinase. Western blotting for β-actin was used as a loading control. Graphs show densitometric analysis of BCATm (B), total BCKD E1{alpha} (C), pS293 E1{alpha} immunoreactivity as a ratio to total BCKD (D), and BCKD E1{alpha} kinase (E). A vertical bar separates the bar graphs for the subcutaneous and visceral depots, because those blots were not run on the same gels, transferred in the same box, or necessarily exposed with the same lot of ECL reagent. Thus comparisons are limited to the samples before and after gastric bypass surgery; a t-test was used to determine significance. *P < 0.05; **P < 0.01; ***P < 0.001, before vs. after surgery. The densitometry values were 8,989 ± 512 before and 9,249 ± 388 after surgery for subcutaneous adipose tissue and 9,683 ± 613 before and 10,046 ± 752 after surgery for visceral adipose tissue (P > 0.05 in both cases).

 
Bariatric surgery in the morbidly obese human subjects led to a roughly 56-kg body weight loss over the course of ~17 mo (Table 5) in both male and female subjects. Average weight loss, plasma glucose, and insulin levels were not significantly different between the male and female subjects by the time of the second operation (Table 5). Because there were no obvious differences in the males and females in terms of these parameters as well as the BCAA metabolizing enzymes or plasma BCAAs, these data were pooled. However, because of the number of tissue samples involved, it was not possible to directly compare visceral and subcutaneous in the before and after conditions. Therefore, each depot was evaluated separately, before and after surgery, using a t-test.


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  		Table 5. Characteristics of human subjects providing visceral and subcutaneous adipose tissue before and after Roux-en-Y gastric bypass surgery

 
Plasma concentrations of BCAA were lower in morbidly obese subjects after gastric bypass, resulting in a significant ~35% decrease in overnight-fasted plasma BCAA concentrations (Fig. 7). The changes in plasma BCAAs were associated with an ~3.5- and ~2-fold increase, respectively, in BCATm in subcutaneous and visceral adipose tissue compared with levels before surgery (Fig. 8, A and B). Total BCKD E1{alpha} was also elevated in both depots (Fig. 8, A and C) compared with that before gastric bypass. No significant changes were observed in BCKD kinase (Fig. 8, A and E), BCKD pS293 phosphorylation/total BCKD ratio (Fig. 8, A and D) in response to weight loss intervention.


   DISCUSSION
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 ABSTRACT
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Obesity-associated hyperaminoacidemia is traditionally interpreted as a consequence of insulin resistance, which affects protein synthesis and proteolysis. However, alterations in the metabolism of the amino acids that we observed could also explain these changes. In the present study we focused on the BCAAs and BCAA catabolism because of the unique obesity/diabetes-related roles of BCAAs (5, 11, 12, 18, 29–31, 59). The potential link between elevations in BCAAs in obesity and the metabolism of BCAAs was examined by comparing effects of obesity and nutritional status changes on BCAAs to changes in either the concentration and/or phosphorylation status of the first two enzymes in BCAA metabolism in key tissues known to be important in BCAA catabolism (liver, skeletal muscle) and in adipose tissue catalyzed by BCATm and BCKD complex.

We found elevated plasma BCAAs in ob/ob mice and high-fat diet-induced obesity in mice and in Zucker fatty rats, in agreement with previous reports in obese humans and Zucker fatty rats (4, 5, 11, 12, 37, 46, 48, 49, 52, 56). These elevations were associated with reductions in the activity or amount of enzymes involved in the early steps of BCAA metabolisms in selective tissues, including liver and fat. BCAAs also declined in morbidly obese humans after surgical weight loss. These changes were comparable to reductions in plasma BCAAs reported upon weight loss by therapeutic starvation (12). In agreement with our findings in rodents, we found that the weight loss-associated declines in plasma BCAAs in humans corresponded to increased concentrations of BCATm and total BCKD E1{alpha} subunit in both visceral and subcutaneous adipose tissues. Finally, in rats and mice, the obesity-related changes in BCAAs tended to normalize after an overnight fast, and in some cases, these changes were mostly reflected in a corresponding statistical change in adipose tissue BCATm and/or BCKD. Thus it is tempting to speculate that changes in adipose tissue mass and its relative contribution to whole body BCAA metabolism may play a role in the elevated BCAAs in obesity.

Rather than being restricted to liver, the BCAA catabolic enzymes are distributed widely in body tissues; and with the exception of the nervous system, all reactions occur in the mitochondria of the cell. The tissue-specific expression and regulation, as well as intracellular compartmentalization, of the branched-chain aminotransferase isozymes (BCATm and BCATc) have an impact on intra- and interorgan exchange of BCAA metabolites, nitrogen cycling, and net nitrogen transfer (54). In lean animals, the liver is thought to be the major organ responsible for oxidative decarboxylation of branched-chain {alpha}-keto acids from the circulation. Whereas BCKD is only partially active because of phosphorylation in other tissues, hepatic BCKD in regularly fed animals is highly active and exhibits low levels of phosphorylation. We found that hepatic BCKD activity was decreased in ob/ob mice and that this was associated with elevated pS293 phosphorylation of the E1{alpha} subunit of BCKD. The differences in phosphorylation state as measured by Western blot analysis with phospho-specific antibodies and BCKD enzyme activity measured in liver suggest that additional proteins and/or factors may modulate BCKD activity in the obese state or that the ratio of free E1 to bound E1 is affected (10, 58). This also could be true in obese fat tissues, where BCKD kinase protein expression was elevated when E1{alpha}-pS293 phosphorylation was unaltered. Hepatic BCKD is highly regulated by dietary protein, physiological factors, and drugs (e.g., clofibrate), largely via alterations of BCKD kinase expression. The active form of hepatic BCKD was significantly decreased by low-protein diets, 48-h starvation, alloxan-diabetes, and once-a-day meal feeding in rats (3, 38, 45). We found that BCKD kinase protein expression in livers and adipose tissues of ob/ob mice and Zucker fatty rats was significantly elevated, potentially explaining the increased S293 phosphorylation of BCKD E1{alpha} subunit. Thyroid hormones also have been shown to induce BCKD kinase expression (41); however, thyroid hormone function is impaired in ob/ob mice (e.g., Ref. 22). Since it has been reported that insulin increased BCKD kinase expression in Clone 9 rat liver cells (42), it is possible that high plasma insulin in obesity may have contributed to the upregulated liver BCKD kinase expression. Although ob/ob mouse liver exhibits severe insulin resistance toward glucose metabolism, it retains insulin sensitivity toward lipid metabolism (41), and this also could be true for the regulation of BCKD kinase expression.

Fat has about twice as much BCATm per milligram of protein than muscle and about five times more dehydrogenase (36). The amount of dehydrogenase is comparable to that in liver (36). Protein levels of BCATm and BCKD E1{alpha} were decreased in adipose tissue from ob/ob mice and Zucker fatty rats. These findings are consistent with studies reporting that mRNA expression for all or some of the adipose tissue enzymes in the BCAA metabolism pathway are decreased in different obesity models (21, 40). It is tempting to speculate that reduction in these BCAA catabolic enzymes may diminish BCAA metabolism in the adipose tissue, but it is hard to quantify how much this would contribute to elevated plasma BCAAs found in ob/ob mice, given that their fat pad weight was many fold greater than that of lean controls. In fat, the drop in BCATm and BCKD E1{alpha} may result in a transient local elevation of BCAA, where BCAAs can act as a nutrient signal to activate mTOR and protein synthesis. Indeed, in randomly fed ob/ob mice, although T389 S6K1 phosphorylation, a common readout of mTOR activation, was not statistically different but quite variable, it was dramatically elevated in adipose tissue compared with lean mice (data not shown). S6K1 hyperphosphorylation could result from both locally elevated BCAAs in fat and hyperinsulinemia in ob/ob mice. Another interesting aspect is that the changes in these enzymes in fat and the plasma BCAAs in obesity seem to be reversible. BCAAs were reduced, whereas BCATm and total E1{alpha} subunit protein levels were elevated, by weight loss in obese humans. Interestingly, changes in plasma BCAAs as well as adipose tissue BCATm, and in Zucker rats even total E1{alpha}, were also impacted by nutritional status, with obesity-related differences dissipating upon fasting. Further studies on roles of BCAAs in adipocyte development, metabolism, and obesity are warranted.

In addition to BCAAs, we noticed changes in other plasma amino acids in ob/ob mice. First, amino acids involved in the urea cycle were also altered. Arg was the only amino acid whose plasma concentration was decreased in the ob/ob mice in the fed state and was further decreased by fasting. Meanwhile, plasma Orn was elevated in both fed and fasted states. The changes in these amino acids could indicate alteration of urea synthesis in the liver, i.e., a block in the cytosolic reactions (increased Orn and Cit) and/or a deficit in Arg from increased metabolism, perhaps for nitric oxide synthesis. Decreased Arg availability could affect vascular dilution in ob/ob mice. Second, in response to fasting, plasma concentrations of most amino acids were decreased in the ob/ob mice but not in the lean mice, consistent with a study in obese Zucker rats (49). Plasma amino acid concentrations represent the balance of protein turnover (protein synthesis and degradation), amino acid absorption from diet, and metabolism of individual amino acids. Obesity could affect all of these factors. In the fed state, elevated food intake and/or decreased protein synthesis (especially in muscle, see above) could contribute to the elevated amino acids, including BCAAs, in plasma. While in the fasted state, protein breakdown in specific tissues could be diminished, leading to lowered plasma amino acids. It also is possible that oxidation of some amino acids was higher in obese animals in the fasted state. A study in obese humans showed that the rate of release of amino acids per unit of forearm and adipose tissue at 22 h of fasting was lower in women with abdominal obesity than in lean women, which may help obese women decrease body protein losses during fasting (44). The lower levels of gluconeogenic amino acids in fasting may be attributed to elevated gluconeogenic rates during fasting (55).

In summary, we have found alterations in BCAA metabolizing enzymes that correspond to obesity-related alterations in plasma BCAA concentrations in fed and fasting obese animal models. The obesity-related rise in BCAAs is seen despite relatively little change in the fed plasma glucose. Fed plasma concentrations of other amino acids were also elevated and largely diminished by fasting in ob/ob mice. Unique alterations in Arg, Orn, and large neutral amino acids including BCAAs were also found in ob/ob mice. Whereas muscle enzymes were unaffected, BCKD kinase expression in liver was increased by obesity, and E1{alpha} S293 phosphorylation was elevated in the face of unaltered BCKD E1{alpha} protein in ob/ob mice and decreased BCKD E1{alpha} protein in Zucker fatty rats. However, these changes remained in the fasted animals when BCAAs normalized. In adipose tissue, obesity was associated with declines in BCATm and BCKD E1{alpha} protein concentrations. These changes were surprisingly dissipated after an overnight fast. It is tempting to speculate therefore that adipose tissue may make a more important contribution to whole body BCAA metabolism than previously realized. Some of the enzymatic alterations in adipose tissue BCAA metabolism were reversed by bariatric surgery-induced weight loss in obese humans, and these were associated with lower plasma BCAA concentrations. Thus the results suggest that tissue-specific alterations in BCAA metabolism could contribute, at least in part, to elevated plasma BCAAs, which may promote protein synthesis to preserve protein loss in the face of insulin resistance in obesity. Since the results of previous studies on BCAA metabolic flux in obesity have been inconsistent, flux studies need to be carefully performed to measure the impact of these enzymatic changes and tissue redistribution of the catabolic enzyme activities on the rates of BCAA metabolism in obesity. In addition, in view of the growing obesity epidemic, the relative importance of adipose tissue as a site of BCAA metabolism and plasma BCAA control should be investigated further.


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 MATERIALS AND METHODS
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DISCLAIMER
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This project was supported by National Institutes of Health Grants DK053843 and DK062880 (to C. J. Lynch) and DK34738 (to S. M. Hutson). The tissue bank is funded, in part, under a grant with the Pennsylvania Department of Health (DOH) using Tobacco Settlement Funds (RNC CM055639-10).


   DISCLAIMER
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 ABSTRACT
 MATERIALS AND METHODS
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REFERENCES
 
The DOH specifically disclaims responsibility for any analyses, interpretations, or conclusions.


   FOOTNOTES
 

Address for reprint requests and other correspondence: C. J. Lynch, Dept. of Cellular and Molecular Physiology, MC-H166, Penn State Univ. 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.


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 RESULTS
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 REFERENCES
 

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