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Adipose Tissue Branched Chain Amino Acid (BCAA) Metabolism Modulates Circulating BCAA Levels *

Adipose Tissue Branched Chain Amino Acid (BCAA) Metabolism Modulates Circulating BCAA Levels * THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 15, pp. 11348 –11356, April 9, 2010 © 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Adipose Tissue Branched Chain Amino Acid (BCAA) □ S Metabolism Modulates Circulating BCAA Levels Received for publication, October 13, 2009, and in revised form, January 18, 2010 Published, JBC Papers in Press, January 21, 2010, DOI 10.1074/jbc.M109.075184 ‡ § ‡ § ‡1 Mark A. Herman , Pengxiang She , Odile D. Peroni , Christopher J. Lynch , and Barbara B. Kahn From the Division of Endocrinology, Diabetes and Metabolism, Beth Israel Deaconess Medical Center, and Department of Medicine, Harvard Medical School, Boston, Massachusetts 02215 and the Department of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033 Whereas the role of adipose tissue in glucose and lipid homeo- BCAAs are poorly metabolized during first pass through the stasis is widely recognized, its role in systemic protein and liver as the liver expresses only low levels of the mitochondrial amino acid metabolism is less well-appreciated. In vitro and ex branched chain aminotransferase (BCAT2 or BCATm), the vivo experiments suggest that adipose tissue can metabolize first enzyme in the catabolism of BCAAs in most peripheral substantial amounts of branched chain amino acids (BCAAs). tissues (2, 3). BCAAs are therefore in a unique position among However, the role of adipose tissue in regulating BCAA metab- amino acids to signal to the periphery and the brain the amino olism in vivo is controversial. Interest in the contribution of adi- acid content of a meal. Circulating BCAAs, acting as nutrient pose tissue to BCAA metabolism has been renewed with recent signals, regulate protein synthesis, and degradation, and insulin observations demonstrating down-regulation of BCAA oxida- secretion, and have been implicated in central nervous system tion enzymes in adipose tissue in obese and insulin-resistant control of food intake and energy balance (4–7). Our knowl- humans. Using gene set enrichment analysis, we observe alter- edge of the physiologic mechanisms which regulate circulating ations in adipose-tissue BCAA enzyme expression caused by BCAA levels remains incomplete. In this study, we provide evi- adipose-selective genetic alterations in the GLUT4 glucose- dence that adipose tissue contributes to the regulation of circu- transporter expression. We show that the rate of adipose tissue lating BCAAs. BCAA oxidation per mg of tissue from normal mice is higher Over the last two decades, adipose tissue has emerged as a than in skeletal muscle. In mice overexpressing GLUT4 specifi- key endocrine organ and a regulator of integrated fuel homeo- cally in adipose tissue, we observe coordinate down-regulation stasis. Whereas its role in glucose and lipid homeostasis is of BCAA metabolizing enzymes selectively in adipose tissue. widely recognized, its role in systemic protein and amino acid This decreases BCAA oxidation rates in adipose tissue, but not metabolism is less well appreciated. However, considerable in in muscle, in association with increased circulating BCAA lev- vitro and ex vivo evidence suggests that adipose tissue is capable els. To confirm the capacity of adipose tissue to modulate circu- of metabolizing significant quantities of BCAAs (8, 9). Based lating BCAA levels in vivo, we demonstrate that transplantation upon ex vivo measurements of leucine flux in rat tissues, of normal adipose tissue into mice that are globally defective in Rosenthal et al. (9) estimated that adipose tissue is second only peripheral BCAA metabolism reduces circulating BCAA levels to skeletal muscle in its capacity to catabolize BCAAs, and that by 30% (fasting)-50% (fed state). These results demonstrate for the capacities of skeletal muscle and adipose tissue are 6–7-fold the first time the capacity of adipose tissue to catabolize circu- larger than liver taking into account the relative masses of lating BCAAs in vivo and that coordinate regulation of adipose- different tissues. However, a study specifically examining the tissue BCAA enzymes may modulate circulating BCAA levels. capacity of the first two enzymes (BCAT2 and the branched chain ketoacid dehydrogenase complex (BCKDHC)) required for BCAA oxidation across different tissues discounted adipose The branched chain amino acids (BCAAs) , leucine, isoleu- tissue as a quantitatively significant site of BCAA catabolism in cine, and valine, are three of the nine essential amino acids and rodents, primates, and humans (10). Similarly, no net uptake of are relatively abundant in the food supply accounting for20% BCAAs could be detected across rat inguinal fat by microdialy- of total protein intake (1). In contrast to the other 17 amino sis sampling or arterio-venous differences (11). Thus the quan- acids, which are predominantly metabolized in the liver, titative significance of adipose tissue BCAA catabolism in vivo was unclear. In the present study, we address this issue. * This work was supported, in whole or in part, by National Institutes of Health Reports of elevated serum BCAA levels (12, 13) and recent Grants R01DK43051 and P30DK57521 (to B. B. K.), Grant K08DK076726 (to observations showing down-regulation of the expression of adi- M. A. H.), and Grant R01DK062880 (to C. J. L.). This work was also supported pose tissue BCAA metabolizing enzymes in obesity and insulin- by a grant from the Picower Foundation (to B. B. K.). □ S The on-line version of this article (available at http://www.jbc.org) contains resistant states (7, 14) has renewed interest in the potential supplemental Fig. S1. significance of adipose tissue BCAA metabolism. Of note, adi- To whom correspondence should be addressed: 330 Brookline Ave., Boston, pose tissue has been hypothesized to be the major site where MA 02215. Tel.: 617-735-3324; Fax: 617-735-3323; E-mail: bkahn@bidmc. harvard.edu. excess BCAAs are stored in the form of lipid (12). Adipose tis- The abbreviations used are: BCAA, branched chain amino acids; BSA, bovine sue efficiently converts BCAA carbon skeletons into newly syn- serum albumin; DTT, dithiothreitol; WT, wild type; GSEA, gene set enrich- thesized fatty acids ex vivo (9). Furthermore, insulin increases ment analysis; BCKDK, branched chain keto acid dehydrogenase kinase; ANOVA, analysis of variance; PMSF, phenylmethylsulfonyl fluoride. the rate of leucine conversion to lipid in adipose tissue explants, 11348 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 15 •APRIL 9, 2010 This is an Open Access article under the CC BY license. Adipose Tissue Modulates BCAA Levels but not in muscle or liver (9). Elevated circulating BCAA levels previously described at 2 months of age (19). Mice were housed and reduced expression of adipose tissue BCAA oxidation singly following surgery and allowed to recover for 2 weeks enzymes in obese individuals normalize following gastric by- prior to further experimentation. Both sham and transplanted pass surgery and weight loss (15). A recent study of monozy- mice were provided free access to both normal chow (NC, Har- gotic twins which were discordant for obesity demonstrated lan 2018) and a purified amino acid BCAA-free diet (-BCAA, down-regulation of the BCAA oxidation enzymes in the obese Dyets 510081). Plasma BCAAs, food intake, and body compo- twin, which correlated with elevated fasting insulin levels and sition were measured 2 weeks after surgery (10 weeks of age). insulin resistance (14). The potential causal relationship be- Mouse studies were conducted in accordance with federal tween altered BCAA enzyme expression and obesity and insu- guidelines and were approved by the Institutional Animal Care lin resistance is of great interest. and Use Committee. We previously made mice with adipose-specific overex- Microarray Analysis—Total RNA from epididymal adipose pression (AG4OX) (16) or knock-out (AG4KO) (17) of the tissue was extracted using the RNeasy Mini Kit from Qiagen insulin-stimulated glucose transporter, GLUT4, because from three mice from each of four genotypes: aP2-Cre trans- GLUT4 is down-regulated selectively in adipose tissue of genic littermates (controls for AG4KO mice), AG4KO mice; obese and type 2 diabetic humans (18). These mice had FVB littermates (controls for AG4OX) and AG4OX. RNA from reciprocal alterations in glucose homeostasis. In the course each mouse was hybridized on an Affymetrix MG-U74-A.v2 of studies to investigate the physiologic mechanisms by Genechip microarray. Affymetrix gene chip hybridization and which GLUT4 expression in adipose tissue regulates sys- analysis were performed at the Genomics Core Facility of the temic fuel homeostasis, we unexpectedly observed coordi- Beth Israel Deaconess Medical Center. Array results were ana- nate down-regulation and up-regulation of branched chain lyzed using DChip software (20). Genome-wide expression amino acid metabolizing enzymes selectively in adipose tis- analysis of the microarray data were performed using gene set sue of AG4OX and AG4KO mice, respectively. In the present enrichment analysis (GSEA) (21). studies, we have taken advantage of the selective down-reg- RNA Extraction and Quantitative Real-time PCR—Tissues ulation of BCAA enzyme expression in AG4OX mice to were harvested from 5-week-old female mice in the fed state (8 examine the physiologic significance of adipose tissue BCAA a.m.), snap frozen in liquid nitrogen, and stored at 80C for oxidation in vivo. We also utilized a second genetic mouse processing. Total RNA was extracted from frozen tissue with model globally defective for peripheral BCAA catabolism to TRI Reagent (Molecular Research Center, Inc.). Real-time PCR investigate whether adipose tissue is capable of modulating was performed using TaqMan One-step RT-PCR Master Mix circulating BCAA levels. Our data show for the first time that (Applied Biosystems) in an Mx3000P thermocycler (Strat- in vivo, adipose tissue can modulate circulating BCAA levels. agene). The Mx3000P software was used to calculate the cycle threshold for each reaction. Relative expression levels were EXPERIMENTAL PROCEDURES determined using the comparative Ct method with normaliza- Animal Studies—Generation and initial metabolic charac- tion of target gene expression levels to 18s. Assay-on-demand terization of the adipose-specific GLUT4-overexpressing (Applied Biosystems) Primer and probe sets are: Bcat2 mice (AG4OX) and adipose-specific GLUT4 knock-out mice Mm00802192_m1;BckdhaMm00476112_m1;DbtMm00501651_ (AG4KO) were previously described (16, 17). Mice were housed m1; Dld Mm00432831_m1; BCKDK Mm00437777_m1. at Beth Israel Deaconess Medical Center with a 14/10 light- Body Composition Analysis—Serial body composition was dark cycle and were fed standard chow (Formulab 5008) ad measured in 7-month-old, female AG4OX mice, and wild-type libitum. All studies were performed on age- and sex-matched littermates by dual-energy x-ray absorptiometry (DEXA) be- littermates. All blood collections were performed by tail vein tween 8 and 10 a.m. on three consecutive days using halo- bleeding. Serial plasma amino acid levels were measured in thane anesthesia. Food was removed following the initial 4-month-old, female AG4OX, and wild-type controls (n  10). body composition measurement and for the subsequent 48 h. Bleeds were performed at baseline (8 AM) and2hand6h Free access to water was provided throughout the fast. For following food removal. For assessment of mTOR signaling, BCAT2 mice, body composition was measured by NMR rapamycin (10 mg/kg body weight, LC Labs) or vehicle (2% (Echo Medical Systems). EtOH in phosphate-buffered saline) was injected intraperitone- Valine Oxidation in Tissue Explants—Valine oxidation and ally in 2-month-old, female AG4OX and wild-type littermates KIV accumulation in tissue explants were measured using a 5 h after food removal. One hour later, insulin (10 units/kg body protocol adapted from Joshi et al. (22). Adipose tissue, soleus, weight) or normal saline was injected via the tail vein. Mice or extensor digitorum longus muscle from fed, 7-month-old, were sacrificed by decapitation 5 min after insulin injection. female AG4OX and wild-type littermates were excised, Plasma was collected, and tissues were harvested, snap frozen weighed, and placed in Erlenmeyer flasks (10 ml) containing 1.5 in liquid nitrogen, and stored at 80C for processing. ml of Krebs-Ringer-phosphate-HEPES buffer (pH 7.4). The BCAT2 mice were housed at Penn State University Col- buffer was supplemented with 5 mM glucose, 1 mM valine con- / 14 lege of Medicine. Prior to surgery male BCAT2 mice were taining 160 Ci/mmol [1- C]valine. For adipose tissue sam- randomized to a sham-operated control group or an adipose ples, the buffer was also supplemented with 200 nM adeno- tissue transplant group (n  6–8 per group). Transplantation sine and 2% (w/v) BSA (fatty acid free). For muscle samples, the of 750 mg of perigonadal fat from wild-type male littermates buffer was supplemented with 0.2% (w/v) BSA. Adipose tissue or sham surgery was performed under halothane anesthesia as samples were minced to 1 mm sized pieces. The flasks were APRIL 9, 2010• VOLUME 285 • NUMBER 15 JOURNAL OF BIOLOGICAL CHEMISTRY 11349 Adipose Tissue Modulates BCAA Levels sealed with rubber stoppers fitted with hanging center wells RESULTS (Kontes, Vineland, NJ) and incubated with shaking at 37 °C. Regulation of BCAA Metabolizing Enzymes in Adipose Tissue— The reaction was terminated after 1 h with injection of 100 lof Initially, we sought to gain insight into the mechanisms by 60% (w/v) perchloric acid into the reaction mixture and 300 l which changes in adipose tissue glucose flux affect adipocyte of 1 M benzothenium hydroxide into the center wells for col- function and systemic fuel metabolism. We performed global lecting the CO produced. After 20 min, the flasks were gene expression analyses on perigonadal adipose tissue from resealed with fresh rubber stoppers fitted with hanging center AG4KO (which are insulin resistant) and AG4OX mice (which wells. Hydrogen peroxide (350 l, 30% w/v) was injected into have enhanced glucose tolerance) (24). Gene set enrichment the reaction mixture and 300 lof1 M benzothenium hydrox- analysis revealed that enzymes of BCAA oxidation are coordi- ide into the center wells for collecting the CO generated by nately down-regulated (supplemental Fig. S1A) and up-regu- the decarboxylation of -ketoisovalerate. lated (supplemental Fig. S1B) in AG4OX and AG4KO adipose Western Blotting—Aliquots of frozen tissues were homoge- tissue, respectively (21). In fact, this was the most highly regu- nized on ice in radioimmunoprecipitation assay buffer supple- lated gene set in adipose tissue of AG4OX and AG4KO mice mented with phospho-preserving and anti-protease agents among 522 gene sets in the data base (data not shown). (sodium fluoride, sodium pyrophosphate, sodium orthovana- BCAT2 catalyzes the reversible, equilibrium conversion of BCAAs to their respective -ketoacids (gene 1, Fig. 1A and B). The date, PMSF, aprotinin, and leupeptin). Protein concentration branched chain ketoacid dehydrogenase complex (BCKDHC) was assayed using the BCA assay. Equal amounts of protein (Fig. 1, A and B, genes 2–5) is a large multisubunit complex that were loaded and transferred to nitrocellulose membranes. The catalyzes the second step, the oxidative decarboxylation of the membranes were probed with antibodies against BCKDHC -ketoacids to their acyl-CoA esters. Decarboxylation is irre- (provided by Dr. Robert Harris), phospho-BCKDH E1 (as pre- versible and considered rate limiting (25). The changes in viously published (23)), p70 S6 kinase (provided by Dr. John expression of BCAA-oxidizing enzymes in adipose tissue of Blenis), and PI3 kinase p85 (Upstate). Results were quantified AG4OX mice are individually relatively small, but are distrib- using the GeneGnome chemiluminescent imaging apparatus uted throughout the pathway and in aggregate are highly statis- and software (Syngene). tically significant by gene set enrichment analysis. Ribosomal S6 Kinase Activity Assay—Tissues were homoge- Q-PCR (Fig. 1C) demonstrated 40–60% reductions in nized in lysis buffer containing 20 mM Tris. pH 7.4, 1% Nonidet BCAT2 and the BCKDHC subunits, BCKDHA and Dbt, in P-40, 10% glycerol, 2 mM EDTA, 10 mM sodium pyrophosphate, AG4OX adipose tissue. Dld, which encodes the dihydrolipoam- 50 mM sodium fluoride, 1 mM DTT, 1 mM sodium orthovana- ide dehydrogenase subunit of the BCDKHC and is shared by the date, 1 mM PMSF, and sigma protease inhibitor mixture. Pro- pyruvate dehydrogenase and -ketoglutarate dehydrogenase tein concentration was determined using the DC protein assay complexes, was unchanged, demonstrating specificity for with BSA as a standard (Bio-Rad). Lysate (200 g) was immu- enzymes unique to the BCAA-metabolizing pathway. BCAA noprecipitated using anti-p70 S6 kinase antibody (provided by enzyme expression in gastrocnemius muscle and liver was Dr. John Blenis) and protein A-Sepharose. Immunoprecipitates unchanged (Fig. 1C). We found that BCAA enzyme expression were washed with 0.5 ml each of lysis buffer, buffer A (1 M NaCl, was higher in perigonadal fat compared with gastrocnemius 10 mM Tris, pH 7.4, 0.1% Nonidet P-40, 2 mM DTT, 1 mM and liver. In liver, BCAT2 expression was very low, consistent phenylmethylsulfonyl fluoride (PMSF)), and buffer B (150 mM with the reported near absence of this enzymatic activity in liver NaCl, 50 mM Tris (pH 7.4, 2 mM DTT, 1 mM PMSF). Kinase (2, 26). activity toward a recombinant GST-S6 peptide (32 final amino Branched chain keto acid dehydrogenase kinase (BCKDK) acids of ribosomal S6) in washed immunoprecipitates was phosphorylates and inhibits the BCKDH complex. Regulation assayed in a reaction containing 200 mM Tris, pH 7.4, 100 mM of BCKDK expression is a key mechanism for nutritional and MgCl , 1 mg/ml BSA, 0.3 g/l GST-S6 peptide, 50 M ATP hormonal regulation of BCAA oxidative flux, particularly in unlabeled, 0.1 Ci [- P]ATP/l reaction volume for 10 min muscle and liver (28). Fig. 1D shows that BCKDK expression in at 30 °C. The reaction was stopped by addition of 2 Laemmli gastrocnemius muscle is6-fold higher than in perigonadal fat buffer containing 200 mM DTT. Reactions were subjected and is nearly undetectable in liver, consistent with prior studies to 18% SDS-PAGE. Gels were stained in Coomassie Blue, indicating that BCKDHC, the target of BCKDK, is largely inac- destained, dried, and the amount of P incorporated into GST- tivated in muscle and nearly completely active in liver of chow- S6 was quantitated by phosphorimaging (Molecular Dynamics fed animals (25). Importantly, BCKDK expression was Storm 860). unchanged (Fig. 1D) in AG4OX perigonadal fat, liver, and mus- Analytical Procedures—Plasma amino acid levels were mea- cle. These results indicate that regulation of BCAA oxidative sured either by HPLC at the Vanderbilt Mouse Metabolic Phe- flux in adipose tissue (see Fig. 2A) can occur independent of notyping Center or by enzymatic assay as previously described changes in BCKDK expression, which is thought to be the clas- (15). sic mechanism by which nutrients and hormones regulate Statistical Analyses—All values are given as means  S.E. BCAA oxidative flux in other tissues. Differences between two groups were assessed using unpaired To determine whether the coordinate changes in BCAA two-tailed Student’s t tests unless otherwise indicated in the enzyme expression result in changes in BCAA oxidation rates, text and figure legends. we measured valine oxidation in tissue explants from AG4OX 11350 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 15 •APRIL 9, 2010 Adipose Tissue Modulates BCAA Levels (30). Although total E1 protein is lower in AG4OX adipose tissue, the ratio of E1 phosphorylation to total E1 is unaltered (Fig. 2B). No changes in total E1 protein, phosphorylated E1, or the ratio of E1 phosphorylation to total E1 were observed in soleus or EDL muscle (Fig. 2, C and D). If BCKDHC is rate limiting for flux through the BCAA oxidation pathway, increased accumulation of the BCKDHC substrate would be expected to accompany a de- crease in BCKDHC activity. How- ever, KIV accumulation was re- duced 4-fold in AG4OX adipose tissue explants compared with con- trol in the absence of IC (Fig. 2A). This suggests that steps upstream of BCKDHC are partially rate-de- termining for valine oxidation in adipose tissue. These results indi- cate a novel mechanism for regula- tion of BCAA oxidation in adipose tissue, i.e. alterations in the expres- sion of BCAA enzymes, in contrast to altered BCKDHC phosphoryla- tion, which is the major mechanism regulating BCAA oxidation in mus- FIGURE 1. Expression of BCAA-oxidizing enzymes in adipose tissue. A, microarray results from adipose cle and liver. tissue from AG4OX versus control and AGKO versus control female mice at 5 weeks of age that were sacrificed Adipose Tissue GLUT4 Overex- in the fed state (n 3 per group). RE, relative expression (the expression in the experimental group relative to pression Regulates Protein Metabo- its control group). B, diagram of the BCAA oxidation pathway indicating genes included in the analysis using the numbering in A. KIC, -ketoisocaproic acid; KIV, -ketoisovaleric acid; KMV, -keto--methylvaleric lism, BCAA Levels, and mTOR Sig- acid. C and D, Q-PCR results for selected enzymes of the BCAA oxidation pathway in perigonadal fat (PG fat), naling—In AGOX mice, the initial gastrocnemius muscle (Gastroc), and liver from fed, 5-week-old female mice. (*, p  0.05 versus WT). fed glucose level is lower and mice and controls. -Chloroisocaproic acid (IC) is an irrevers- blood glucose falls more rapidly 2 and 6 h after food removal ible inhibitor of BCKDK leading to rapid dephosphorylation (Fig. 3A). Insulin levels are not different between groups at and activation of BCKDHC (29). Valine oxidation in the any time point (Fig. 3A). Given the susceptibility to fasting absence of IC reflects basal valine oxidation whereas IC elic- hypoglycemia in AG4OX mice and the importance of mobi- its maximal stimulation of valine oxidation. Compared with lization of fat and protein to defend against hypoglycemia, controls, basal, and maximally stimulated valine oxidation was we investigated whether prolonged fasting leads to changes 6.6- and 4-fold lower, respectively, in perigonadal adipose tis- in whole-body composition in this model. AG4OX mice are sue from AG4OX mice (Fig. 2A). The basal rate of valine oxi- modestly obese compared with wild-type littermates (Fig. dation in adipose tissue per mg of wet tissue weight was 2-fold 3B). AG4OX mice lost significantly more weight than con- greater than in oxidative muscle (soleus) and 6-fold greater trols during a 48 h fast (Fig. 3C). Loss of lean mass accounted than in glycolytic muscle (EDL). We do not see regulation of for all of the excess weight loss in AG4OX mice. Control valine oxidation in either soleus or EDL muscle of AG4OX mice animals maintained their percent lean mass (Fig. 3D) and except for a small decrease in soleus in the presence of IC. percent lipid mass at constant levels throughout the fast. In Even taking into account the relative amounts of adipose and contrast, in AG4OX mice, lean mass decreased (from 68 to muscle mass in vivo, these results suggest that adipose tissue 64% (p  0.001)) and lipid mass increased (from 34% to 37% may contribute a quantitatively significant amount to whole (p  0.001)) as percentages of total body mass. Thus, over- body BCAA oxidation. expression of GLUT4 specifically in adipose tissue resulted We sought to determine whether BCAA metabolism under- in derangements in systemic fuel metabolism associated goes the same regulation in adipose tissue as in muscle and with preservation of fat mass at the expense of lean body liver. Phosphorylation of the E1 subunit of BCKHDC by mass during a prolonged fast. BCKDK has been reported to be the major site for nutritional or We next asked whether the decreases in BCAA enzyme hormonal regulation of BCKDHC activity and BCAA oxidation expression in adipose tissue and the increased protein degrada- APRIL 9, 2010• VOLUME 285 • NUMBER 15 JOURNAL OF BIOLOGICAL CHEMISTRY 11351 Adipose Tissue Modulates BCAA Levels FIGURE 2. Valine oxidation and E1 phosphorylation in skeletal muscle and adipose tissue. A, valine oxidation and KIV accumulation were mea- sured with and without addition of -chloroisocaproic acid (IC) in adipose tissue (n 10 per group), soleus (n 6 per group), and EDL (n 6 per group) explants from fed, 7-month-old female AG4OX and wild-type control mice. Comparisons within tissues performed by 2-way ANOVA using Tukey’s test for post-hoc analysis between groups; *, p  0.05 comparing effect of geno- type within IC; #, p  0.05 comparing effect of IC within genotype; Com- parison across tissues in wild-type animals without IC performed by one- way ANOVA, ‡, p 0.05. B and C, quantitation of Western blots for total BKDH E1, phospho-BCKDH, and the ratio of total to phospho-BCKDH E1 for (B) perigonadal fat with representative blot, and for (C) soleus and (D) EDL mus- cle from tissues harvested in experiment described in A. (n 5– 6 per group, *, p  0.05 versus WT by t test). tion during fasting in AG4OX affect circulating BCAAs. Levels of all three BCAAs were elevated in the fed state and 2 and 6 h following food removal (Fig. 3E). If this were due to increased net protein breakdown as indicated by the greater loss of lean FIGURE 3. Changes in glycemia, protein metabolism, and circulating amino mass in AG4OX, levels of other circulating amino acids should acid levels following food removal. A, serial glucose and insulin levels were measured in 4-month-old, female AG4OX and wild-type controls (n  10 per also be elevated (31). We were surprised to find no increase in group). Tail vein bleeds were performed at baseline (8 a.m.) and2hand6h the circulating levels of other essential amino acids including following food removal (*, p 0.05 for WT versus AG4OX at each time). B–D, serial body composition measurements were made by DEXA in 7-month-old female phenylalanine, threonine, and methionine in AG4OX (Fig. 3F). AG4OX and wild-type control mice at baseline and after 24 h and 48 h of fasting Because changes in net protein breakdown affect most amino (n 9 per group). Comparisons were made for changes in (B) total body weight, acids proportionately, the specific increases in circulating lipid weight, lean weight (C) total cumulative weight loss, and (D) % lean mass and % lipid mass (E, WT, f, AG4OX). Body weight, lipid weight, and lean weight BCAA levels suggest a selective decrease in BCAA clearance differed between genotypes (*, p  0.05). Body weight, lipid weight, and lean (31). Taken together with the selective decrease in BCAA oxi- weight differed within genotypes at 24 and 48 h of fasting compared with time 0 dation in adipose tissue in AG4OX mice and the high rate of (p 0.05, paired Student’s t test). % lean mass and % lipid mass differed in AG4OX mice after 24 and 48 h of fasting compared with time 0 (p 0.05, paired Student’s BCAA oxidation in adipose tissue explants compared with skel- t test), but remained unchanged in wild-type mice. E and F, serial plasma amino etal muscle, these data suggest that adipose tissue is an impor- acid levels were measured under the conditions described in A (*, p 0.05 for WT versus AG4OX at each time). tant site for whole body BCAA oxidation and thereby affects 11352 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 15 •APRIL 9, 2010 Adipose Tissue Modulates BCAA Levels nounced in adipose tissue of AG4OX mice (Fig. 4B). In con- trast, insulin stimulated a similar degree of mobility shift in gastrocnemius of AG4OX compared with WT mice. Pre- treatment with rapamycin prevented the insulin-stimulated shift in S6 Kinase gel mobility in both WT and AG4OX mice in both tissues. The increase in circulating BCAAs may con- tribute to the increase in adipose tissue insulin-stimulated S6 kinase activity in AG4OX mice or may be the direct result of increased glucose flux to enhance mTOR signaling. How- ever, these results indicate that the increase in circulating BCAAs in fasted AG4OX mice are not sufficient to increase insulin stimulated mTOR activation in other insulin target tissues. Fat Transplantation Reduces BCAAs in Mice Defective for Peripheral BCAA Metabolism—Mice lacking BCAT2 have massively elevated circulating BCAA levels due to the inability to oxidize BCAAs in peripheral tissues, and they are hyper- metabolic (7). To determine whether adipose tissue BCAA oxidation is sufficient to affect systemic BCAA homeostasis and circulating BCAA levels, we transplanted 750 mg of adipose tissue from wild-type littermates into BCAT2 mice. BCAT2 mice were randomized to sham surgery or trans- plant. Prior to surgery, body weights (sham: 23.5  0.6 g versus transplant: 23.4  0.9; p  0.92) and fed BCAA levels (sham: 7.0 1.4 mM versus transplant: 6.7 0.9; p 0.86) were similar between the two groups of BCAT2 mice. Two weeks after transplantation or sham surgery (Fig. 5A), body weight and body composition were not different between the two groups. Transplantation of wild-type fat into BCAT2 mice lowered fed plasma BCAAs 46% and BCAAs 6 h after food removal 31% compared with sham-operated BCAT2 controls (Fig. 5B). Fed plasma alanine levels increased 58% after transplantation of wild-type fat into BCAT2 mice (Fig. 5C). Fed plasma gluta- mine levels tended to increase (16%) in the transplanted mice as well. The increase in circulating alanine and tendency toward FIGURE 4. Changes in mTOR signaling. A, p70 S6 kinase activity in perigo- increased glutamine may reflect increased BCAA oxidation nadal adipose tissue, liver, gastrocnemius muscle, and tibialis anterior muscle in the transplanted adipose tissue since adipose tissue can of 2-month-old female, WT, and AG4OX mice. Awake mice were injected with utilize BCAA-derived nitrogen to synthesize alanine and saline or insulin (10 units/kg ip) and sacrificed 5 min later (n 7–9 per group). Tissues were frozen for assays. Statistical comparisons in performed by 2-way glutamine (8, 32). ANOVA with Tukey’s post-hoc testing; *, p  0.001 for insulin effect within Both sham and transplanted mice were provided free genotype; †, p  0.001 compared with WT insulin group; &, p  0.057 com- pared with WT-saline group. B, representative blot demonstrating p70 S6 access to normal chow and a BCAA-free diet to prevent tox- kinase gel mobility shift measured by Western blotting in perigonadal fat and icity associated with extreme elevations in plasma BCAAs in gastrocnemius muscle in animals described in A. Mice were pretreated with BCAT2 mice (7). Intake of total food, normal chow, or rapamycin (10 mg/kg ip) or vehicle 1 h before saline or insulin injection. BCAA-free diet (Fig. 5D) was not different though a trend circulating BCAA levels. Changes in BCAA oxidation in other toward increased BCAA-free diet intake was noted in the tissues could also contribute. transplanted group (p  0.057). Plasma BCAAs in the trans- Because circulating BCAAs have been implicated as nutri- planted group tend to be lower than in the sham-operated ent signals, we examined the mTOR signaling pathway in the group for any amount of normal chow intake (Fig. 5E). The AG4OX model. There was a tendency for a 2-fold stimula- ratio of normal chow (g/day) to plasma BCAA levels tends to tion of S6 kinase (S6K) activity by insulin in adipose tissue of be higher in the transplanted group (0.34  0.12) compared WT mice (Fig. 4A). In AG4OX, basal (saline injection) S6K with the sham-operated group (0.20  0.04, p  0.13). Thus, activity was the same as WT. Strikingly, insulin stimulated the reduction in circulating BCAAs in the transplant group S6K activity 6.5-fold in WAT of AG4OX mice. There was no does not result from reduced BCAA intake but is due to increase in S6K activity in liver, gastrocnemius muscle, or increased BCAA clearance, most likely through catabolism tibialis anterior muscle (Fig. 4A) of AG4OX compared with in the transplanted normal fat. The reductions in plasma WT. Insulin injection in WT mice induced a gel mobility BCAAs in the transplant group occurred in the absence of shift in S6K in adipose tissue and, consistent with the S6K changes in fed or fasted glycemia, insulin or leptin levels activity, the insulin-stimulated shift in S6K was more pro- (Table 1). These results strongly support the conclusion that APRIL 9, 2010• VOLUME 285 • NUMBER 15 JOURNAL OF BIOLOGICAL CHEMISTRY 11353 Adipose Tissue Modulates BCAA Levels GSEA of adipose tissue from mice with adipose-selective alter- ations in Glut4 expression. In AG4OX mice, down-regulation of the BCAA oxidative enzymes caused a significant decrease in BCAA oxidation in adipose tissue explants. This decrease could contribute to the increased circulating BCAA levels in AG4OX mice. Results from the AG4OX mice provide insights into the mechanisms by which BCAA oxidative flux may be regu- lated. Activity of BCKDHC in muscle and liver is potently regulated by inhibitory phosphorylation by BCKDK in response to nutritional and hormonal cues (30). The dramatic alterations in BCAA oxidation rates in AG4OX adipose tissue explants occurred independently of changes in BCKDK expression and BCKDHC phosphorylation suggesting that there may be important differences in the mechanisms regulating BCAA metabolism in adipose tissue compared with muscle and liver. Numerous investigators have considered BCKDHC to be the rate-determining enzyme in BCAA oxidation (33–37). Accord- ing to metabolic control theory, if BCKDHC were rate-deter- mining, down-regulation of its activity would be expected to result in accumulation of its substrates (38). Despite the signif- icant reductions in BCKDHC expression in AG4OX adipose tissue, the accumulation of KIV decreased 4-fold in AG4OX adipose tissue explants compared with controls. Thus, in adi- pose tissue explants, BCKDHC does not appear to be rate-lim- iting. Consistent with modern metabolic control theory (38) and as has been documented for other metabolic pathways such as the fatty acid synthesis pathway (39), small but coordinate changes in the expression level of enzymes distributed through- out a pathway can translate into substantial changes in the rate of flux through that pathway. FIGURE 5. BCAT2 fat transplantation. A and B, body weight, body composi- tion, and plasma BCAAs in the fed state and following6hof food removal The results from the AG4OX mice provide additional in- were measured in male BCAT2 mice transplanted with 750 mg of wild- sights into the mechanisms by which BCAA oxidative flux may type fat versus sham operated controls (n  6 – 8 per group; *, p  0.05). C, plasma alanine and glutamine were measured in the fed mice described be regulated. We observe a profound decrease in adipose tissue above (n  4 –7 per group; *, p  0.05). Mice had free access to a choice of BCAA oxidation as a result of adipose-specific GLUT4 overex- normal chow (NC) or BCAA-free diet and food intake was measured for 1 week pression. Our results might suggest that the increased glucose at 10 weeks of age, following the 2 week recovery period. D, food intake is presented as average daily values. E, fed plasma BCAA levels for individual flux in AG4OX adipocytes can impair BCAA oxidative flux per mice versus average daily intake of normal chow. se. However, treating adipose tissue ex vivo with glucose either with or without insulin increases rather than decreases BCAA oxidation rates acutely (8, 40). Glucose likely exerts its positive TABLE 1 effect on BCAA oxidation by increasing the availability of co- Fed and fasting blood metabolites after fat transplantation Three weeks after surgery, blood was collected from 8 to 10 a.m. from sham-oper- factors required for transamination and/or decarboxylation ated controls and fat-transplanted Bcat2 mice in the fed state and after an over- (41). However, our study now demonstrates that the coordi- night fast. Values are means S.E. n 6–8 per group. Statistical significance at a p value of 0.05 was not achieved for any comparison between sham control and fat nate down-regulation of BCAA oxidative enzyme expression transplant groups. and resulting decrease in BCAA oxidative flux is dominant Sham control Fat transplant over the positive effects of glucose to increase BCAA oxida- Fed Fasted Fed Fasted tive flux. Glucose (mg/dl) 216  16 97  9 186 488  2 It is of interest to use these ex vivo measurements to estimate Insulin (ng/ml) 0.95  0.02 0.21  0.01 0.82  0.16 0.19  0.03 the potential contribution of adipose tissue BCAA oxidation to Leptin (ng/ml) 1.58  0.32 0.37  0.12 2.29  0.29 0.41  0.06 Glucose-insulin 204  15 20  2.5 151  26 16  2.1 whole body BCAA oxidation. In our wild-type mice, adipose product tissue weighs 7.5 g and lean mass weighs 20.8 g. Assuming that skeletal muscle accounts for the majority of lean mass and in vivo, adipose tissue is an important site of BCAA oxidation extrapolating our ex vivo measurements of BCAA flux averag- and is capable of contributing significantly to regulation of ing the soleus and Edl measurements to obtain a representative circulating BCAA levels. skeletal muscle flux, adipose tissue could account for oxidation DISCUSSION of 950 nmol BCAAs/hour comparable to the 830 nmol We observed the coordinate and reciprocal regulation of BCAAs/hour in skeletal muscle. Despite the modestly in- expression of enzymes involved in BCAA oxidation using creased adiposity in AG4OX mice (10.8 g), the BCAA oxidation 11354 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 15 •APRIL 9, 2010 Adipose Tissue Modulates BCAA Levels rate would remain 80% lower in AG4OX (205 nmol/h) com- vation negatively feeds back on the insulin signaling pathway pared with control at the whole adipose tissue organ level. via inhibitory serine phosphorylation of IRS1 by S6K (45, 46). In These estimates must be interpreted with the large caveats our study, insulin-stimulated S6K activity in AG4OX adi- that other organs such as liver, kidney, and brain are pose tissue is markedly increased compared with controls, included in the lean mass measurement. Additionally, we but activity in muscle and liver is not different from controls. assume that the oxidation rate in perigonadal fat is repre- These results agree with other studies that have shown that sentative of all fat pads and the average of soleus and Edl are increased circulating leucine alone is insufficient to increase representative of all skeletal muscle. Lastly, these calcula- mTOR signaling in all tissues (47). In AG4OX adipose tissue, tions are based upon ex vivo measurements in which sub- either the increased glucose flux or the increased circulating strate availability is constant. In vivo, the actual flux rates BCAAs may contribute to the increased insulin-stimulated may be significantly affected by differences in blood flow and SK6 activity. delivery of substrate to different tissues. Whereas the physiologic role of adipose tissue BCAA oxi- Although calculated estimates of adipose tissue’s capacity for dation remains uncertain, our observations support the con- BCAA oxidation indicate that adipose tissue BCAA metabo- clusion that the coordinate down-regulation of BCAA oxi- lism may be physiologically significant, experimental evidence dative enzymes may dramatically alter adipose tissue BCAA confirming this in vivo are lacking. Ex vivo, rat epididymal fat oxidative flux. This is of interest because down-regulation of pads release glutamine and alanine in response to increasing adipose tissue BCAA oxidative enzymes has also recently been BCAA exposure (8, 32) and BCAAs were hypothesized to pro- observed in obese humans and the expression inversely corre- vide the nitrogen for net alanine and glutamine synthesis. Arte- lates with insulin-resistance (14, 15, 48). Adipose tissue BCAA riovenous sampling across rat inguinal fat pads and human sub- enzyme expression increases with surgically-induced weight cutaneous fat pads confirmed net alanine and glutamine release loss (15) or thiazolidinedione treatment (48) and parallels from adipose tissue, and Frayn et al. (11, 42) suggested that improvements in insulin sensitivity. In addition, in an unbiased BCAAs were the most likely source of nitrogen. A study in metabolomics-based profiling approach, an elevated circulat- anesthetized rats failed to detect arteriovenous differences in ing BCAA-related metabolic “signature” best predicted insulin- BCAA concentrations across the inguinal fat pad although it resistance in human subjects (13). was suggested that BCAAs derived from intracellular proteol- The down-regulation of BCAA oxidative enzymes in AG4OX ysis were catabolized in adipose tissue and contributed to glu- mice may provide a new perspective on the physiologic signif- tamine synthesis and release (11). Our data now demonstrate icance of adipose tissue BCAA oxidation. Physiologic states in that in vivo, adipose tissue can avidly metabolize circulating which the capacity of adipose tissue to catabolize BCAAs falls BCAAs at least when BCAA levels are markedly elevated. We rapidly are fasting (8, 27, 49) or feeding a protein-deficient diet show that transplanting only 750 mg of fat (less than 10–20% of (8). The rapid decrease in adipose tissue capacity to degrade total fat mass in a lean mouse) from wild-type mice into BCAAs with fasting has been suggested to preserve BCAAs for BCAT2 mice is sufficient to dramatically lower circulating glucose production or ketogenesis and prevent their irreversi- BCAA levels. Furthermore, plasma alanine levels increase after ble conversion to lipids for storage (27). There is indirect sup- transplantation consistent with the potential role of adipose port for this in a study suggesting that the proportion of radio- tissue BCAA oxidation to provide a nitrogenous source for ala- labeled leucine carbon skeletons stored as lipid decreased with nine (and/or glutamine) synthesis. Insulin can reduce circulat- fasting (8). The down-regulation of BCAA oxidation in AG4OX ing BCAAs (43), but the reduction in plasma BCAAs in our adipose tissue may represent a physiological adaptation to pro- transplant study occurs in the absence of any changes in circu- tect against fasting hypoglycemia (Fig. 3) by preserving BCAA lating insulin. Increased BCAA catabolism in the transplanted carbon for gluconeogenesis and ketogenesis in the liver rather fat most likely accounts for the dramatic decrease in circulating than lipogenesis in adipose tissue. BCAAs in the transplanted mice though we cannot exclude Goodman and Frick (41) suggested that the down-regulation indirect effects of fat transplantation to impact BCAA metabo- of BCAA enzymes with fasting or protein deficiency may be lism in other tissues. However, these results conclusively dem- signaled by decreased insulin or leucine per se. In the AG4OX onstrate for the first time the potential for adipose tissue to alter mice, coordinate down-regulation of the adipose tissue circulating BCAA levels in vivo. BCAA oxidation enzymes occurs despite the fact that circu- The marked elevations in circulating BCAAs in BCAT2 lating leucine and insulin levels are increased or normal. mice have profound effects on growth, metabolism, and viabil- Thus alterations in plasma insulin or leucine levels cannot ity (7). It is not surprising that we did not observe normalization explain the regulation of BCAA oxidative enzymes in this of these metabolic parameters in the transplanted BCAT2 model although this does not exclude the possibility of a mice because despite a 30–50% reduction in circulating BCAA novel circulating factor. levels with transplantation, the circulating BCAA levels remain In conclusion, the present study demonstrates the poten- severalfold higher than in wild-type mice. tial capacity for adipose tissue to regulate circulating BCAAs Additional putative consequences of elevated circulating BCAAs are through mTOR signaling either as a nutrient signal in vivo. Further it shows an important relationship in vivo to increase protein synthesis or by contributing to down-regu- between regulation of adipose glucose and BCAA metabo- lation of insulin signaling. Leucine in synergy with insulin acti- lism. 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Adipose Tissue Branched Chain Amino Acid (BCAA) Metabolism Modulates Circulating BCAA Levels *

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American Society for Biochemistry and Molecular Biology
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Copyright © 2010 Elsevier Inc.
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0021-9258
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10.1074/jbc.m109.075184
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Abstract

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 15, pp. 11348 –11356, April 9, 2010 © 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Adipose Tissue Branched Chain Amino Acid (BCAA) □ S Metabolism Modulates Circulating BCAA Levels Received for publication, October 13, 2009, and in revised form, January 18, 2010 Published, JBC Papers in Press, January 21, 2010, DOI 10.1074/jbc.M109.075184 ‡ § ‡ § ‡1 Mark A. Herman , Pengxiang She , Odile D. Peroni , Christopher J. Lynch , and Barbara B. Kahn From the Division of Endocrinology, Diabetes and Metabolism, Beth Israel Deaconess Medical Center, and Department of Medicine, Harvard Medical School, Boston, Massachusetts 02215 and the Department of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033 Whereas the role of adipose tissue in glucose and lipid homeo- BCAAs are poorly metabolized during first pass through the stasis is widely recognized, its role in systemic protein and liver as the liver expresses only low levels of the mitochondrial amino acid metabolism is less well-appreciated. In vitro and ex branched chain aminotransferase (BCAT2 or BCATm), the vivo experiments suggest that adipose tissue can metabolize first enzyme in the catabolism of BCAAs in most peripheral substantial amounts of branched chain amino acids (BCAAs). tissues (2, 3). BCAAs are therefore in a unique position among However, the role of adipose tissue in regulating BCAA metab- amino acids to signal to the periphery and the brain the amino olism in vivo is controversial. Interest in the contribution of adi- acid content of a meal. Circulating BCAAs, acting as nutrient pose tissue to BCAA metabolism has been renewed with recent signals, regulate protein synthesis, and degradation, and insulin observations demonstrating down-regulation of BCAA oxida- secretion, and have been implicated in central nervous system tion enzymes in adipose tissue in obese and insulin-resistant control of food intake and energy balance (4–7). Our knowl- humans. Using gene set enrichment analysis, we observe alter- edge of the physiologic mechanisms which regulate circulating ations in adipose-tissue BCAA enzyme expression caused by BCAA levels remains incomplete. In this study, we provide evi- adipose-selective genetic alterations in the GLUT4 glucose- dence that adipose tissue contributes to the regulation of circu- transporter expression. We show that the rate of adipose tissue lating BCAAs. BCAA oxidation per mg of tissue from normal mice is higher Over the last two decades, adipose tissue has emerged as a than in skeletal muscle. In mice overexpressing GLUT4 specifi- key endocrine organ and a regulator of integrated fuel homeo- cally in adipose tissue, we observe coordinate down-regulation stasis. Whereas its role in glucose and lipid homeostasis is of BCAA metabolizing enzymes selectively in adipose tissue. widely recognized, its role in systemic protein and amino acid This decreases BCAA oxidation rates in adipose tissue, but not metabolism is less well appreciated. However, considerable in in muscle, in association with increased circulating BCAA lev- vitro and ex vivo evidence suggests that adipose tissue is capable els. To confirm the capacity of adipose tissue to modulate circu- of metabolizing significant quantities of BCAAs (8, 9). Based lating BCAA levels in vivo, we demonstrate that transplantation upon ex vivo measurements of leucine flux in rat tissues, of normal adipose tissue into mice that are globally defective in Rosenthal et al. (9) estimated that adipose tissue is second only peripheral BCAA metabolism reduces circulating BCAA levels to skeletal muscle in its capacity to catabolize BCAAs, and that by 30% (fasting)-50% (fed state). These results demonstrate for the capacities of skeletal muscle and adipose tissue are 6–7-fold the first time the capacity of adipose tissue to catabolize circu- larger than liver taking into account the relative masses of lating BCAAs in vivo and that coordinate regulation of adipose- different tissues. However, a study specifically examining the tissue BCAA enzymes may modulate circulating BCAA levels. capacity of the first two enzymes (BCAT2 and the branched chain ketoacid dehydrogenase complex (BCKDHC)) required for BCAA oxidation across different tissues discounted adipose The branched chain amino acids (BCAAs) , leucine, isoleu- tissue as a quantitatively significant site of BCAA catabolism in cine, and valine, are three of the nine essential amino acids and rodents, primates, and humans (10). Similarly, no net uptake of are relatively abundant in the food supply accounting for20% BCAAs could be detected across rat inguinal fat by microdialy- of total protein intake (1). In contrast to the other 17 amino sis sampling or arterio-venous differences (11). Thus the quan- acids, which are predominantly metabolized in the liver, titative significance of adipose tissue BCAA catabolism in vivo was unclear. In the present study, we address this issue. * This work was supported, in whole or in part, by National Institutes of Health Reports of elevated serum BCAA levels (12, 13) and recent Grants R01DK43051 and P30DK57521 (to B. B. K.), Grant K08DK076726 (to observations showing down-regulation of the expression of adi- M. A. H.), and Grant R01DK062880 (to C. J. L.). This work was also supported pose tissue BCAA metabolizing enzymes in obesity and insulin- by a grant from the Picower Foundation (to B. B. K.). □ S The on-line version of this article (available at http://www.jbc.org) contains resistant states (7, 14) has renewed interest in the potential supplemental Fig. S1. significance of adipose tissue BCAA metabolism. Of note, adi- To whom correspondence should be addressed: 330 Brookline Ave., Boston, pose tissue has been hypothesized to be the major site where MA 02215. Tel.: 617-735-3324; Fax: 617-735-3323; E-mail: bkahn@bidmc. harvard.edu. excess BCAAs are stored in the form of lipid (12). Adipose tis- The abbreviations used are: BCAA, branched chain amino acids; BSA, bovine sue efficiently converts BCAA carbon skeletons into newly syn- serum albumin; DTT, dithiothreitol; WT, wild type; GSEA, gene set enrich- thesized fatty acids ex vivo (9). Furthermore, insulin increases ment analysis; BCKDK, branched chain keto acid dehydrogenase kinase; ANOVA, analysis of variance; PMSF, phenylmethylsulfonyl fluoride. the rate of leucine conversion to lipid in adipose tissue explants, 11348 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 15 •APRIL 9, 2010 This is an Open Access article under the CC BY license. Adipose Tissue Modulates BCAA Levels but not in muscle or liver (9). Elevated circulating BCAA levels previously described at 2 months of age (19). Mice were housed and reduced expression of adipose tissue BCAA oxidation singly following surgery and allowed to recover for 2 weeks enzymes in obese individuals normalize following gastric by- prior to further experimentation. Both sham and transplanted pass surgery and weight loss (15). A recent study of monozy- mice were provided free access to both normal chow (NC, Har- gotic twins which were discordant for obesity demonstrated lan 2018) and a purified amino acid BCAA-free diet (-BCAA, down-regulation of the BCAA oxidation enzymes in the obese Dyets 510081). Plasma BCAAs, food intake, and body compo- twin, which correlated with elevated fasting insulin levels and sition were measured 2 weeks after surgery (10 weeks of age). insulin resistance (14). The potential causal relationship be- Mouse studies were conducted in accordance with federal tween altered BCAA enzyme expression and obesity and insu- guidelines and were approved by the Institutional Animal Care lin resistance is of great interest. and Use Committee. We previously made mice with adipose-specific overex- Microarray Analysis—Total RNA from epididymal adipose pression (AG4OX) (16) or knock-out (AG4KO) (17) of the tissue was extracted using the RNeasy Mini Kit from Qiagen insulin-stimulated glucose transporter, GLUT4, because from three mice from each of four genotypes: aP2-Cre trans- GLUT4 is down-regulated selectively in adipose tissue of genic littermates (controls for AG4KO mice), AG4KO mice; obese and type 2 diabetic humans (18). These mice had FVB littermates (controls for AG4OX) and AG4OX. RNA from reciprocal alterations in glucose homeostasis. In the course each mouse was hybridized on an Affymetrix MG-U74-A.v2 of studies to investigate the physiologic mechanisms by Genechip microarray. Affymetrix gene chip hybridization and which GLUT4 expression in adipose tissue regulates sys- analysis were performed at the Genomics Core Facility of the temic fuel homeostasis, we unexpectedly observed coordi- Beth Israel Deaconess Medical Center. Array results were ana- nate down-regulation and up-regulation of branched chain lyzed using DChip software (20). Genome-wide expression amino acid metabolizing enzymes selectively in adipose tis- analysis of the microarray data were performed using gene set sue of AG4OX and AG4KO mice, respectively. In the present enrichment analysis (GSEA) (21). studies, we have taken advantage of the selective down-reg- RNA Extraction and Quantitative Real-time PCR—Tissues ulation of BCAA enzyme expression in AG4OX mice to were harvested from 5-week-old female mice in the fed state (8 examine the physiologic significance of adipose tissue BCAA a.m.), snap frozen in liquid nitrogen, and stored at 80C for oxidation in vivo. We also utilized a second genetic mouse processing. Total RNA was extracted from frozen tissue with model globally defective for peripheral BCAA catabolism to TRI Reagent (Molecular Research Center, Inc.). Real-time PCR investigate whether adipose tissue is capable of modulating was performed using TaqMan One-step RT-PCR Master Mix circulating BCAA levels. Our data show for the first time that (Applied Biosystems) in an Mx3000P thermocycler (Strat- in vivo, adipose tissue can modulate circulating BCAA levels. agene). The Mx3000P software was used to calculate the cycle threshold for each reaction. Relative expression levels were EXPERIMENTAL PROCEDURES determined using the comparative Ct method with normaliza- Animal Studies—Generation and initial metabolic charac- tion of target gene expression levels to 18s. Assay-on-demand terization of the adipose-specific GLUT4-overexpressing (Applied Biosystems) Primer and probe sets are: Bcat2 mice (AG4OX) and adipose-specific GLUT4 knock-out mice Mm00802192_m1;BckdhaMm00476112_m1;DbtMm00501651_ (AG4KO) were previously described (16, 17). Mice were housed m1; Dld Mm00432831_m1; BCKDK Mm00437777_m1. at Beth Israel Deaconess Medical Center with a 14/10 light- Body Composition Analysis—Serial body composition was dark cycle and were fed standard chow (Formulab 5008) ad measured in 7-month-old, female AG4OX mice, and wild-type libitum. All studies were performed on age- and sex-matched littermates by dual-energy x-ray absorptiometry (DEXA) be- littermates. All blood collections were performed by tail vein tween 8 and 10 a.m. on three consecutive days using halo- bleeding. Serial plasma amino acid levels were measured in thane anesthesia. Food was removed following the initial 4-month-old, female AG4OX, and wild-type controls (n  10). body composition measurement and for the subsequent 48 h. Bleeds were performed at baseline (8 AM) and2hand6h Free access to water was provided throughout the fast. For following food removal. For assessment of mTOR signaling, BCAT2 mice, body composition was measured by NMR rapamycin (10 mg/kg body weight, LC Labs) or vehicle (2% (Echo Medical Systems). EtOH in phosphate-buffered saline) was injected intraperitone- Valine Oxidation in Tissue Explants—Valine oxidation and ally in 2-month-old, female AG4OX and wild-type littermates KIV accumulation in tissue explants were measured using a 5 h after food removal. One hour later, insulin (10 units/kg body protocol adapted from Joshi et al. (22). Adipose tissue, soleus, weight) or normal saline was injected via the tail vein. Mice or extensor digitorum longus muscle from fed, 7-month-old, were sacrificed by decapitation 5 min after insulin injection. female AG4OX and wild-type littermates were excised, Plasma was collected, and tissues were harvested, snap frozen weighed, and placed in Erlenmeyer flasks (10 ml) containing 1.5 in liquid nitrogen, and stored at 80C for processing. ml of Krebs-Ringer-phosphate-HEPES buffer (pH 7.4). The BCAT2 mice were housed at Penn State University Col- buffer was supplemented with 5 mM glucose, 1 mM valine con- / 14 lege of Medicine. Prior to surgery male BCAT2 mice were taining 160 Ci/mmol [1- C]valine. For adipose tissue sam- randomized to a sham-operated control group or an adipose ples, the buffer was also supplemented with 200 nM adeno- tissue transplant group (n  6–8 per group). Transplantation sine and 2% (w/v) BSA (fatty acid free). For muscle samples, the of 750 mg of perigonadal fat from wild-type male littermates buffer was supplemented with 0.2% (w/v) BSA. Adipose tissue or sham surgery was performed under halothane anesthesia as samples were minced to 1 mm sized pieces. The flasks were APRIL 9, 2010• VOLUME 285 • NUMBER 15 JOURNAL OF BIOLOGICAL CHEMISTRY 11349 Adipose Tissue Modulates BCAA Levels sealed with rubber stoppers fitted with hanging center wells RESULTS (Kontes, Vineland, NJ) and incubated with shaking at 37 °C. Regulation of BCAA Metabolizing Enzymes in Adipose Tissue— The reaction was terminated after 1 h with injection of 100 lof Initially, we sought to gain insight into the mechanisms by 60% (w/v) perchloric acid into the reaction mixture and 300 l which changes in adipose tissue glucose flux affect adipocyte of 1 M benzothenium hydroxide into the center wells for col- function and systemic fuel metabolism. We performed global lecting the CO produced. After 20 min, the flasks were gene expression analyses on perigonadal adipose tissue from resealed with fresh rubber stoppers fitted with hanging center AG4KO (which are insulin resistant) and AG4OX mice (which wells. Hydrogen peroxide (350 l, 30% w/v) was injected into have enhanced glucose tolerance) (24). Gene set enrichment the reaction mixture and 300 lof1 M benzothenium hydrox- analysis revealed that enzymes of BCAA oxidation are coordi- ide into the center wells for collecting the CO generated by nately down-regulated (supplemental Fig. S1A) and up-regu- the decarboxylation of -ketoisovalerate. lated (supplemental Fig. S1B) in AG4OX and AG4KO adipose Western Blotting—Aliquots of frozen tissues were homoge- tissue, respectively (21). In fact, this was the most highly regu- nized on ice in radioimmunoprecipitation assay buffer supple- lated gene set in adipose tissue of AG4OX and AG4KO mice mented with phospho-preserving and anti-protease agents among 522 gene sets in the data base (data not shown). (sodium fluoride, sodium pyrophosphate, sodium orthovana- BCAT2 catalyzes the reversible, equilibrium conversion of BCAAs to their respective -ketoacids (gene 1, Fig. 1A and B). The date, PMSF, aprotinin, and leupeptin). Protein concentration branched chain ketoacid dehydrogenase complex (BCKDHC) was assayed using the BCA assay. Equal amounts of protein (Fig. 1, A and B, genes 2–5) is a large multisubunit complex that were loaded and transferred to nitrocellulose membranes. The catalyzes the second step, the oxidative decarboxylation of the membranes were probed with antibodies against BCKDHC -ketoacids to their acyl-CoA esters. Decarboxylation is irre- (provided by Dr. Robert Harris), phospho-BCKDH E1 (as pre- versible and considered rate limiting (25). The changes in viously published (23)), p70 S6 kinase (provided by Dr. John expression of BCAA-oxidizing enzymes in adipose tissue of Blenis), and PI3 kinase p85 (Upstate). Results were quantified AG4OX mice are individually relatively small, but are distrib- using the GeneGnome chemiluminescent imaging apparatus uted throughout the pathway and in aggregate are highly statis- and software (Syngene). tically significant by gene set enrichment analysis. Ribosomal S6 Kinase Activity Assay—Tissues were homoge- Q-PCR (Fig. 1C) demonstrated 40–60% reductions in nized in lysis buffer containing 20 mM Tris. pH 7.4, 1% Nonidet BCAT2 and the BCKDHC subunits, BCKDHA and Dbt, in P-40, 10% glycerol, 2 mM EDTA, 10 mM sodium pyrophosphate, AG4OX adipose tissue. Dld, which encodes the dihydrolipoam- 50 mM sodium fluoride, 1 mM DTT, 1 mM sodium orthovana- ide dehydrogenase subunit of the BCDKHC and is shared by the date, 1 mM PMSF, and sigma protease inhibitor mixture. Pro- pyruvate dehydrogenase and -ketoglutarate dehydrogenase tein concentration was determined using the DC protein assay complexes, was unchanged, demonstrating specificity for with BSA as a standard (Bio-Rad). Lysate (200 g) was immu- enzymes unique to the BCAA-metabolizing pathway. BCAA noprecipitated using anti-p70 S6 kinase antibody (provided by enzyme expression in gastrocnemius muscle and liver was Dr. John Blenis) and protein A-Sepharose. Immunoprecipitates unchanged (Fig. 1C). We found that BCAA enzyme expression were washed with 0.5 ml each of lysis buffer, buffer A (1 M NaCl, was higher in perigonadal fat compared with gastrocnemius 10 mM Tris, pH 7.4, 0.1% Nonidet P-40, 2 mM DTT, 1 mM and liver. In liver, BCAT2 expression was very low, consistent phenylmethylsulfonyl fluoride (PMSF)), and buffer B (150 mM with the reported near absence of this enzymatic activity in liver NaCl, 50 mM Tris (pH 7.4, 2 mM DTT, 1 mM PMSF). Kinase (2, 26). activity toward a recombinant GST-S6 peptide (32 final amino Branched chain keto acid dehydrogenase kinase (BCKDK) acids of ribosomal S6) in washed immunoprecipitates was phosphorylates and inhibits the BCKDH complex. Regulation assayed in a reaction containing 200 mM Tris, pH 7.4, 100 mM of BCKDK expression is a key mechanism for nutritional and MgCl , 1 mg/ml BSA, 0.3 g/l GST-S6 peptide, 50 M ATP hormonal regulation of BCAA oxidative flux, particularly in unlabeled, 0.1 Ci [- P]ATP/l reaction volume for 10 min muscle and liver (28). Fig. 1D shows that BCKDK expression in at 30 °C. The reaction was stopped by addition of 2 Laemmli gastrocnemius muscle is6-fold higher than in perigonadal fat buffer containing 200 mM DTT. Reactions were subjected and is nearly undetectable in liver, consistent with prior studies to 18% SDS-PAGE. Gels were stained in Coomassie Blue, indicating that BCKDHC, the target of BCKDK, is largely inac- destained, dried, and the amount of P incorporated into GST- tivated in muscle and nearly completely active in liver of chow- S6 was quantitated by phosphorimaging (Molecular Dynamics fed animals (25). Importantly, BCKDK expression was Storm 860). unchanged (Fig. 1D) in AG4OX perigonadal fat, liver, and mus- Analytical Procedures—Plasma amino acid levels were mea- cle. These results indicate that regulation of BCAA oxidative sured either by HPLC at the Vanderbilt Mouse Metabolic Phe- flux in adipose tissue (see Fig. 2A) can occur independent of notyping Center or by enzymatic assay as previously described changes in BCKDK expression, which is thought to be the clas- (15). sic mechanism by which nutrients and hormones regulate Statistical Analyses—All values are given as means  S.E. BCAA oxidative flux in other tissues. Differences between two groups were assessed using unpaired To determine whether the coordinate changes in BCAA two-tailed Student’s t tests unless otherwise indicated in the enzyme expression result in changes in BCAA oxidation rates, text and figure legends. we measured valine oxidation in tissue explants from AG4OX 11350 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 15 •APRIL 9, 2010 Adipose Tissue Modulates BCAA Levels (30). Although total E1 protein is lower in AG4OX adipose tissue, the ratio of E1 phosphorylation to total E1 is unaltered (Fig. 2B). No changes in total E1 protein, phosphorylated E1, or the ratio of E1 phosphorylation to total E1 were observed in soleus or EDL muscle (Fig. 2, C and D). If BCKDHC is rate limiting for flux through the BCAA oxidation pathway, increased accumulation of the BCKDHC substrate would be expected to accompany a de- crease in BCKDHC activity. How- ever, KIV accumulation was re- duced 4-fold in AG4OX adipose tissue explants compared with con- trol in the absence of IC (Fig. 2A). This suggests that steps upstream of BCKDHC are partially rate-de- termining for valine oxidation in adipose tissue. These results indi- cate a novel mechanism for regula- tion of BCAA oxidation in adipose tissue, i.e. alterations in the expres- sion of BCAA enzymes, in contrast to altered BCKDHC phosphoryla- tion, which is the major mechanism regulating BCAA oxidation in mus- FIGURE 1. Expression of BCAA-oxidizing enzymes in adipose tissue. A, microarray results from adipose cle and liver. tissue from AG4OX versus control and AGKO versus control female mice at 5 weeks of age that were sacrificed Adipose Tissue GLUT4 Overex- in the fed state (n 3 per group). RE, relative expression (the expression in the experimental group relative to pression Regulates Protein Metabo- its control group). B, diagram of the BCAA oxidation pathway indicating genes included in the analysis using the numbering in A. KIC, -ketoisocaproic acid; KIV, -ketoisovaleric acid; KMV, -keto--methylvaleric lism, BCAA Levels, and mTOR Sig- acid. C and D, Q-PCR results for selected enzymes of the BCAA oxidation pathway in perigonadal fat (PG fat), naling—In AGOX mice, the initial gastrocnemius muscle (Gastroc), and liver from fed, 5-week-old female mice. (*, p  0.05 versus WT). fed glucose level is lower and mice and controls. -Chloroisocaproic acid (IC) is an irrevers- blood glucose falls more rapidly 2 and 6 h after food removal ible inhibitor of BCKDK leading to rapid dephosphorylation (Fig. 3A). Insulin levels are not different between groups at and activation of BCKDHC (29). Valine oxidation in the any time point (Fig. 3A). Given the susceptibility to fasting absence of IC reflects basal valine oxidation whereas IC elic- hypoglycemia in AG4OX mice and the importance of mobi- its maximal stimulation of valine oxidation. Compared with lization of fat and protein to defend against hypoglycemia, controls, basal, and maximally stimulated valine oxidation was we investigated whether prolonged fasting leads to changes 6.6- and 4-fold lower, respectively, in perigonadal adipose tis- in whole-body composition in this model. AG4OX mice are sue from AG4OX mice (Fig. 2A). The basal rate of valine oxi- modestly obese compared with wild-type littermates (Fig. dation in adipose tissue per mg of wet tissue weight was 2-fold 3B). AG4OX mice lost significantly more weight than con- greater than in oxidative muscle (soleus) and 6-fold greater trols during a 48 h fast (Fig. 3C). Loss of lean mass accounted than in glycolytic muscle (EDL). We do not see regulation of for all of the excess weight loss in AG4OX mice. Control valine oxidation in either soleus or EDL muscle of AG4OX mice animals maintained their percent lean mass (Fig. 3D) and except for a small decrease in soleus in the presence of IC. percent lipid mass at constant levels throughout the fast. In Even taking into account the relative amounts of adipose and contrast, in AG4OX mice, lean mass decreased (from 68 to muscle mass in vivo, these results suggest that adipose tissue 64% (p  0.001)) and lipid mass increased (from 34% to 37% may contribute a quantitatively significant amount to whole (p  0.001)) as percentages of total body mass. Thus, over- body BCAA oxidation. expression of GLUT4 specifically in adipose tissue resulted We sought to determine whether BCAA metabolism under- in derangements in systemic fuel metabolism associated goes the same regulation in adipose tissue as in muscle and with preservation of fat mass at the expense of lean body liver. Phosphorylation of the E1 subunit of BCKHDC by mass during a prolonged fast. BCKDK has been reported to be the major site for nutritional or We next asked whether the decreases in BCAA enzyme hormonal regulation of BCKDHC activity and BCAA oxidation expression in adipose tissue and the increased protein degrada- APRIL 9, 2010• VOLUME 285 • NUMBER 15 JOURNAL OF BIOLOGICAL CHEMISTRY 11351 Adipose Tissue Modulates BCAA Levels FIGURE 2. Valine oxidation and E1 phosphorylation in skeletal muscle and adipose tissue. A, valine oxidation and KIV accumulation were mea- sured with and without addition of -chloroisocaproic acid (IC) in adipose tissue (n 10 per group), soleus (n 6 per group), and EDL (n 6 per group) explants from fed, 7-month-old female AG4OX and wild-type control mice. Comparisons within tissues performed by 2-way ANOVA using Tukey’s test for post-hoc analysis between groups; *, p  0.05 comparing effect of geno- type within IC; #, p  0.05 comparing effect of IC within genotype; Com- parison across tissues in wild-type animals without IC performed by one- way ANOVA, ‡, p 0.05. B and C, quantitation of Western blots for total BKDH E1, phospho-BCKDH, and the ratio of total to phospho-BCKDH E1 for (B) perigonadal fat with representative blot, and for (C) soleus and (D) EDL mus- cle from tissues harvested in experiment described in A. (n 5– 6 per group, *, p  0.05 versus WT by t test). tion during fasting in AG4OX affect circulating BCAAs. Levels of all three BCAAs were elevated in the fed state and 2 and 6 h following food removal (Fig. 3E). If this were due to increased net protein breakdown as indicated by the greater loss of lean FIGURE 3. Changes in glycemia, protein metabolism, and circulating amino mass in AG4OX, levels of other circulating amino acids should acid levels following food removal. A, serial glucose and insulin levels were measured in 4-month-old, female AG4OX and wild-type controls (n  10 per also be elevated (31). We were surprised to find no increase in group). Tail vein bleeds were performed at baseline (8 a.m.) and2hand6h the circulating levels of other essential amino acids including following food removal (*, p 0.05 for WT versus AG4OX at each time). B–D, serial body composition measurements were made by DEXA in 7-month-old female phenylalanine, threonine, and methionine in AG4OX (Fig. 3F). AG4OX and wild-type control mice at baseline and after 24 h and 48 h of fasting Because changes in net protein breakdown affect most amino (n 9 per group). Comparisons were made for changes in (B) total body weight, acids proportionately, the specific increases in circulating lipid weight, lean weight (C) total cumulative weight loss, and (D) % lean mass and % lipid mass (E, WT, f, AG4OX). Body weight, lipid weight, and lean weight BCAA levels suggest a selective decrease in BCAA clearance differed between genotypes (*, p  0.05). Body weight, lipid weight, and lean (31). Taken together with the selective decrease in BCAA oxi- weight differed within genotypes at 24 and 48 h of fasting compared with time 0 dation in adipose tissue in AG4OX mice and the high rate of (p 0.05, paired Student’s t test). % lean mass and % lipid mass differed in AG4OX mice after 24 and 48 h of fasting compared with time 0 (p 0.05, paired Student’s BCAA oxidation in adipose tissue explants compared with skel- t test), but remained unchanged in wild-type mice. E and F, serial plasma amino etal muscle, these data suggest that adipose tissue is an impor- acid levels were measured under the conditions described in A (*, p 0.05 for WT versus AG4OX at each time). tant site for whole body BCAA oxidation and thereby affects 11352 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 15 •APRIL 9, 2010 Adipose Tissue Modulates BCAA Levels nounced in adipose tissue of AG4OX mice (Fig. 4B). In con- trast, insulin stimulated a similar degree of mobility shift in gastrocnemius of AG4OX compared with WT mice. Pre- treatment with rapamycin prevented the insulin-stimulated shift in S6 Kinase gel mobility in both WT and AG4OX mice in both tissues. The increase in circulating BCAAs may con- tribute to the increase in adipose tissue insulin-stimulated S6 kinase activity in AG4OX mice or may be the direct result of increased glucose flux to enhance mTOR signaling. How- ever, these results indicate that the increase in circulating BCAAs in fasted AG4OX mice are not sufficient to increase insulin stimulated mTOR activation in other insulin target tissues. Fat Transplantation Reduces BCAAs in Mice Defective for Peripheral BCAA Metabolism—Mice lacking BCAT2 have massively elevated circulating BCAA levels due to the inability to oxidize BCAAs in peripheral tissues, and they are hyper- metabolic (7). To determine whether adipose tissue BCAA oxidation is sufficient to affect systemic BCAA homeostasis and circulating BCAA levels, we transplanted 750 mg of adipose tissue from wild-type littermates into BCAT2 mice. BCAT2 mice were randomized to sham surgery or trans- plant. Prior to surgery, body weights (sham: 23.5  0.6 g versus transplant: 23.4  0.9; p  0.92) and fed BCAA levels (sham: 7.0 1.4 mM versus transplant: 6.7 0.9; p 0.86) were similar between the two groups of BCAT2 mice. Two weeks after transplantation or sham surgery (Fig. 5A), body weight and body composition were not different between the two groups. Transplantation of wild-type fat into BCAT2 mice lowered fed plasma BCAAs 46% and BCAAs 6 h after food removal 31% compared with sham-operated BCAT2 controls (Fig. 5B). Fed plasma alanine levels increased 58% after transplantation of wild-type fat into BCAT2 mice (Fig. 5C). Fed plasma gluta- mine levels tended to increase (16%) in the transplanted mice as well. The increase in circulating alanine and tendency toward FIGURE 4. Changes in mTOR signaling. A, p70 S6 kinase activity in perigo- increased glutamine may reflect increased BCAA oxidation nadal adipose tissue, liver, gastrocnemius muscle, and tibialis anterior muscle in the transplanted adipose tissue since adipose tissue can of 2-month-old female, WT, and AG4OX mice. Awake mice were injected with utilize BCAA-derived nitrogen to synthesize alanine and saline or insulin (10 units/kg ip) and sacrificed 5 min later (n 7–9 per group). Tissues were frozen for assays. Statistical comparisons in performed by 2-way glutamine (8, 32). ANOVA with Tukey’s post-hoc testing; *, p  0.001 for insulin effect within Both sham and transplanted mice were provided free genotype; †, p  0.001 compared with WT insulin group; &, p  0.057 com- pared with WT-saline group. B, representative blot demonstrating p70 S6 access to normal chow and a BCAA-free diet to prevent tox- kinase gel mobility shift measured by Western blotting in perigonadal fat and icity associated with extreme elevations in plasma BCAAs in gastrocnemius muscle in animals described in A. Mice were pretreated with BCAT2 mice (7). Intake of total food, normal chow, or rapamycin (10 mg/kg ip) or vehicle 1 h before saline or insulin injection. BCAA-free diet (Fig. 5D) was not different though a trend circulating BCAA levels. Changes in BCAA oxidation in other toward increased BCAA-free diet intake was noted in the tissues could also contribute. transplanted group (p  0.057). Plasma BCAAs in the trans- Because circulating BCAAs have been implicated as nutri- planted group tend to be lower than in the sham-operated ent signals, we examined the mTOR signaling pathway in the group for any amount of normal chow intake (Fig. 5E). The AG4OX model. There was a tendency for a 2-fold stimula- ratio of normal chow (g/day) to plasma BCAA levels tends to tion of S6 kinase (S6K) activity by insulin in adipose tissue of be higher in the transplanted group (0.34  0.12) compared WT mice (Fig. 4A). In AG4OX, basal (saline injection) S6K with the sham-operated group (0.20  0.04, p  0.13). Thus, activity was the same as WT. Strikingly, insulin stimulated the reduction in circulating BCAAs in the transplant group S6K activity 6.5-fold in WAT of AG4OX mice. There was no does not result from reduced BCAA intake but is due to increase in S6K activity in liver, gastrocnemius muscle, or increased BCAA clearance, most likely through catabolism tibialis anterior muscle (Fig. 4A) of AG4OX compared with in the transplanted normal fat. The reductions in plasma WT. Insulin injection in WT mice induced a gel mobility BCAAs in the transplant group occurred in the absence of shift in S6K in adipose tissue and, consistent with the S6K changes in fed or fasted glycemia, insulin or leptin levels activity, the insulin-stimulated shift in S6K was more pro- (Table 1). These results strongly support the conclusion that APRIL 9, 2010• VOLUME 285 • NUMBER 15 JOURNAL OF BIOLOGICAL CHEMISTRY 11353 Adipose Tissue Modulates BCAA Levels GSEA of adipose tissue from mice with adipose-selective alter- ations in Glut4 expression. In AG4OX mice, down-regulation of the BCAA oxidative enzymes caused a significant decrease in BCAA oxidation in adipose tissue explants. This decrease could contribute to the increased circulating BCAA levels in AG4OX mice. Results from the AG4OX mice provide insights into the mechanisms by which BCAA oxidative flux may be regu- lated. Activity of BCKDHC in muscle and liver is potently regulated by inhibitory phosphorylation by BCKDK in response to nutritional and hormonal cues (30). The dramatic alterations in BCAA oxidation rates in AG4OX adipose tissue explants occurred independently of changes in BCKDK expression and BCKDHC phosphorylation suggesting that there may be important differences in the mechanisms regulating BCAA metabolism in adipose tissue compared with muscle and liver. Numerous investigators have considered BCKDHC to be the rate-determining enzyme in BCAA oxidation (33–37). Accord- ing to metabolic control theory, if BCKDHC were rate-deter- mining, down-regulation of its activity would be expected to result in accumulation of its substrates (38). Despite the signif- icant reductions in BCKDHC expression in AG4OX adipose tissue, the accumulation of KIV decreased 4-fold in AG4OX adipose tissue explants compared with controls. Thus, in adi- pose tissue explants, BCKDHC does not appear to be rate-lim- iting. Consistent with modern metabolic control theory (38) and as has been documented for other metabolic pathways such as the fatty acid synthesis pathway (39), small but coordinate changes in the expression level of enzymes distributed through- out a pathway can translate into substantial changes in the rate of flux through that pathway. FIGURE 5. BCAT2 fat transplantation. A and B, body weight, body composi- tion, and plasma BCAAs in the fed state and following6hof food removal The results from the AG4OX mice provide additional in- were measured in male BCAT2 mice transplanted with 750 mg of wild- sights into the mechanisms by which BCAA oxidative flux may type fat versus sham operated controls (n  6 – 8 per group; *, p  0.05). C, plasma alanine and glutamine were measured in the fed mice described be regulated. We observe a profound decrease in adipose tissue above (n  4 –7 per group; *, p  0.05). Mice had free access to a choice of BCAA oxidation as a result of adipose-specific GLUT4 overex- normal chow (NC) or BCAA-free diet and food intake was measured for 1 week pression. Our results might suggest that the increased glucose at 10 weeks of age, following the 2 week recovery period. D, food intake is presented as average daily values. E, fed plasma BCAA levels for individual flux in AG4OX adipocytes can impair BCAA oxidative flux per mice versus average daily intake of normal chow. se. However, treating adipose tissue ex vivo with glucose either with or without insulin increases rather than decreases BCAA oxidation rates acutely (8, 40). Glucose likely exerts its positive TABLE 1 effect on BCAA oxidation by increasing the availability of co- Fed and fasting blood metabolites after fat transplantation Three weeks after surgery, blood was collected from 8 to 10 a.m. from sham-oper- factors required for transamination and/or decarboxylation ated controls and fat-transplanted Bcat2 mice in the fed state and after an over- (41). However, our study now demonstrates that the coordi- night fast. Values are means S.E. n 6–8 per group. Statistical significance at a p value of 0.05 was not achieved for any comparison between sham control and fat nate down-regulation of BCAA oxidative enzyme expression transplant groups. and resulting decrease in BCAA oxidative flux is dominant Sham control Fat transplant over the positive effects of glucose to increase BCAA oxida- Fed Fasted Fed Fasted tive flux. Glucose (mg/dl) 216  16 97  9 186 488  2 It is of interest to use these ex vivo measurements to estimate Insulin (ng/ml) 0.95  0.02 0.21  0.01 0.82  0.16 0.19  0.03 the potential contribution of adipose tissue BCAA oxidation to Leptin (ng/ml) 1.58  0.32 0.37  0.12 2.29  0.29 0.41  0.06 Glucose-insulin 204  15 20  2.5 151  26 16  2.1 whole body BCAA oxidation. In our wild-type mice, adipose product tissue weighs 7.5 g and lean mass weighs 20.8 g. Assuming that skeletal muscle accounts for the majority of lean mass and in vivo, adipose tissue is an important site of BCAA oxidation extrapolating our ex vivo measurements of BCAA flux averag- and is capable of contributing significantly to regulation of ing the soleus and Edl measurements to obtain a representative circulating BCAA levels. skeletal muscle flux, adipose tissue could account for oxidation DISCUSSION of 950 nmol BCAAs/hour comparable to the 830 nmol We observed the coordinate and reciprocal regulation of BCAAs/hour in skeletal muscle. Despite the modestly in- expression of enzymes involved in BCAA oxidation using creased adiposity in AG4OX mice (10.8 g), the BCAA oxidation 11354 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 15 •APRIL 9, 2010 Adipose Tissue Modulates BCAA Levels rate would remain 80% lower in AG4OX (205 nmol/h) com- vation negatively feeds back on the insulin signaling pathway pared with control at the whole adipose tissue organ level. via inhibitory serine phosphorylation of IRS1 by S6K (45, 46). In These estimates must be interpreted with the large caveats our study, insulin-stimulated S6K activity in AG4OX adi- that other organs such as liver, kidney, and brain are pose tissue is markedly increased compared with controls, included in the lean mass measurement. Additionally, we but activity in muscle and liver is not different from controls. assume that the oxidation rate in perigonadal fat is repre- These results agree with other studies that have shown that sentative of all fat pads and the average of soleus and Edl are increased circulating leucine alone is insufficient to increase representative of all skeletal muscle. Lastly, these calcula- mTOR signaling in all tissues (47). In AG4OX adipose tissue, tions are based upon ex vivo measurements in which sub- either the increased glucose flux or the increased circulating strate availability is constant. In vivo, the actual flux rates BCAAs may contribute to the increased insulin-stimulated may be significantly affected by differences in blood flow and SK6 activity. delivery of substrate to different tissues. Whereas the physiologic role of adipose tissue BCAA oxi- Although calculated estimates of adipose tissue’s capacity for dation remains uncertain, our observations support the con- BCAA oxidation indicate that adipose tissue BCAA metabo- clusion that the coordinate down-regulation of BCAA oxi- lism may be physiologically significant, experimental evidence dative enzymes may dramatically alter adipose tissue BCAA confirming this in vivo are lacking. Ex vivo, rat epididymal fat oxidative flux. This is of interest because down-regulation of pads release glutamine and alanine in response to increasing adipose tissue BCAA oxidative enzymes has also recently been BCAA exposure (8, 32) and BCAAs were hypothesized to pro- observed in obese humans and the expression inversely corre- vide the nitrogen for net alanine and glutamine synthesis. Arte- lates with insulin-resistance (14, 15, 48). Adipose tissue BCAA riovenous sampling across rat inguinal fat pads and human sub- enzyme expression increases with surgically-induced weight cutaneous fat pads confirmed net alanine and glutamine release loss (15) or thiazolidinedione treatment (48) and parallels from adipose tissue, and Frayn et al. (11, 42) suggested that improvements in insulin sensitivity. In addition, in an unbiased BCAAs were the most likely source of nitrogen. A study in metabolomics-based profiling approach, an elevated circulat- anesthetized rats failed to detect arteriovenous differences in ing BCAA-related metabolic “signature” best predicted insulin- BCAA concentrations across the inguinal fat pad although it resistance in human subjects (13). was suggested that BCAAs derived from intracellular proteol- The down-regulation of BCAA oxidative enzymes in AG4OX ysis were catabolized in adipose tissue and contributed to glu- mice may provide a new perspective on the physiologic signif- tamine synthesis and release (11). Our data now demonstrate icance of adipose tissue BCAA oxidation. Physiologic states in that in vivo, adipose tissue can avidly metabolize circulating which the capacity of adipose tissue to catabolize BCAAs falls BCAAs at least when BCAA levels are markedly elevated. We rapidly are fasting (8, 27, 49) or feeding a protein-deficient diet show that transplanting only 750 mg of fat (less than 10–20% of (8). The rapid decrease in adipose tissue capacity to degrade total fat mass in a lean mouse) from wild-type mice into BCAAs with fasting has been suggested to preserve BCAAs for BCAT2 mice is sufficient to dramatically lower circulating glucose production or ketogenesis and prevent their irreversi- BCAA levels. Furthermore, plasma alanine levels increase after ble conversion to lipids for storage (27). There is indirect sup- transplantation consistent with the potential role of adipose port for this in a study suggesting that the proportion of radio- tissue BCAA oxidation to provide a nitrogenous source for ala- labeled leucine carbon skeletons stored as lipid decreased with nine (and/or glutamine) synthesis. Insulin can reduce circulat- fasting (8). The down-regulation of BCAA oxidation in AG4OX ing BCAAs (43), but the reduction in plasma BCAAs in our adipose tissue may represent a physiological adaptation to pro- transplant study occurs in the absence of any changes in circu- tect against fasting hypoglycemia (Fig. 3) by preserving BCAA lating insulin. Increased BCAA catabolism in the transplanted carbon for gluconeogenesis and ketogenesis in the liver rather fat most likely accounts for the dramatic decrease in circulating than lipogenesis in adipose tissue. BCAAs in the transplanted mice though we cannot exclude Goodman and Frick (41) suggested that the down-regulation indirect effects of fat transplantation to impact BCAA metabo- of BCAA enzymes with fasting or protein deficiency may be lism in other tissues. However, these results conclusively dem- signaled by decreased insulin or leucine per se. In the AG4OX onstrate for the first time the potential for adipose tissue to alter mice, coordinate down-regulation of the adipose tissue circulating BCAA levels in vivo. BCAA oxidation enzymes occurs despite the fact that circu- The marked elevations in circulating BCAAs in BCAT2 lating leucine and insulin levels are increased or normal. mice have profound effects on growth, metabolism, and viabil- Thus alterations in plasma insulin or leucine levels cannot ity (7). It is not surprising that we did not observe normalization explain the regulation of BCAA oxidative enzymes in this of these metabolic parameters in the transplanted BCAT2 model although this does not exclude the possibility of a mice because despite a 30–50% reduction in circulating BCAA novel circulating factor. levels with transplantation, the circulating BCAA levels remain In conclusion, the present study demonstrates the poten- severalfold higher than in wild-type mice. tial capacity for adipose tissue to regulate circulating BCAAs Additional putative consequences of elevated circulating BCAAs are through mTOR signaling either as a nutrient signal in vivo. Further it shows an important relationship in vivo to increase protein synthesis or by contributing to down-regu- between regulation of adipose glucose and BCAA metabo- lation of insulin signaling. Leucine in synergy with insulin acti- lism. 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Journal

Journal of Biological ChemistryAmerican Society for Biochemistry and Molecular Biology

Published: Apr 9, 2010

Keywords: Diseases/Diabetes; Diseases/Metabolic; Metabolism/Amino Acid; Metabolism/Carbohydrate; Adipose tissue metabolism; Fasting; Integrated Physiology; mTOR

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