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Inflammation and ER Stress Regulate Branched-ChainAmino Acid Uptake and Metabolism in Adipocytes

Joel S. Burrill, Eric K. Long, Brian Reilly, Yingfeng Deng, Ian M. Armitage,Philipp E. Scherer, and David A. Bernlohr

Department of Biochemistry, Molecular Biology, and Biophysics (J.S.B., E.K.L., B.R., I.M.A., D.A.B.),University of Minnesota-Twin Cities, Minneapolis, Minnesota 55455; and Touchstone Diabetes Center(Y.D., P.E.S.) and Department of Internal Medicine (Y.D., P.E.S.), University of Texas SouthwesternMedical Center, Dallas, Texas 75390

Inflammation plays a critical role in the pathology of obesity-linked insulin resistance and ismechanistically linked to the effects of macrophage-derived cytokines on adipocyte energy me-tabolism, particularly that of the mitochondrial branched-chain amino acid (BCAA) and tricarbox-ylic acid (TCA) pathways. To address the role of inflammation on energy metabolism in adipocytes,we used high fat-fed C57BL/6J mice and lean controls and measured the down-regulation of geneslinked to BCAA and TCA cycle metabolism selectively in visceral but not in subcutaneous adiposetissue, brown fat, liver, or muscle. Using 3T3-L1 cells, TNF�, and other proinflammatory cytokinetreatments reduced the expression of the genes linked to BCAA transport and oxidation. Consis-tent with this, [14C]-leucine uptake and conversion to triglycerides was markedly attenuated inTNF�-treated adipocytes, whereas the conversion to protein was relatively unaffected. Becauseinflammatory cytokines lead to the induction of endoplasmic reticulum stress, we evaluated theeffects of tunicamycin or thapsigargin treatment of 3T3-L1 cells and measured a similar down-regulation in the BCAA/TCA cycle pathway. Moreover, transgenic mice overexpressing X-boxbinding protein 1 in adipocytes similarly down-regulated genes of BCAA and TCA metabolism invivo. These results indicate that inflammation and endoplasmic reticulum stress attenuate lipo-genesis in visceral adipose depots by down-regulating the BCAA/TCA metabolism pathway andare consistent with a model whereby the accumulation of serum BCAA in the obese insulin-resistant state is linked to adipose inflammation. (Molecular Endocrinology 29: 411–420, 2015)

Obesity-linked type 2 diabetes and its associatedhealth complications are major health care concerns

worldwide and at the molecular level are linked to in-creased abundance and activity of classically activatedmacrophages and other immune cells in adipose tissue(1–3). Increased immune cells produce a chronic low-grade inflammatory state exemplified by elevated adiposeTNF�, IL-6, IL-1�, and interferon (IFN)-� primarily invisceral, and to a lesser extent, subcutaneous depots (4).The positive inflammatory poise of adipose tissue leads tothe down-regulation of adipocyte genes linked to the an-tiinflammatory response system and an increase in mito-chondrial reactive oxygen species (ROS), increased endo-

plasmic reticulum (ER) stress, and loss of electrontransport activity (3, 5). Although the exact molecularmechanisms that tie ROS to insulin resistance are com-plex, the ROS-dependent activation of the unfolded pro-tein response and the nuclear factor-�B (NF-�B)/c-JunN-terminal kinase signaling pathways have been impli-cated by a variety of in situ and in vivo models (6–8).

Recent work has identified adipose tissue and adi-pocytes as a major contributor to whole-body, branched-chain amino acid catabolism (9). The branched-chainamino acids leucine, isoleucine, and valine are trans-ported into adipocytes and metabolized in the mitochon-dria to form the anapleurotic intermediates acetyl-CoA

ISSN Print 0888-8809 ISSN Online 1944-9917Printed in U.S.A.Copyright © 2015 by the Endocrine SocietyReceived August 27, 2014. Accepted January 26, 2015.First Published Online January 30, 2015

Abbreviations: BCAA, branched-chain amino acid; Bcat2, branched-chain amino-acidtransaminase 2; ER, endoplasmic reticulum; IFN, interferon; NF-�B, nuclear factor-�B;NMR, nuclear magnetic resonance; ROS, reactive oxygen species; Sdha, succinate dehy-drogenase �-subunit; TCA, tricarboxylic acid; Xbp1, X-box binding protein 1.

O R I G I N A L R E S E A R C H

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and succinyl-CoA, thereby enabling maximal pyruvatemetabolism to citrate and subsequent lipogenesis (10).Work by Herman et al. (9) have shown that adipocytesreadily metabolize branched-chain amino acid (BCAA) tolipogenic precursors and that the maximal lipogenic raterequires replenishment of the TCA cycle. Recentlymetabolomic studies of human obesity and metabolicdysfunction have revealed a connection between circulat-ing BCAAs and insulin resistance, and a variety of anal-yses have demonstrated that the accumulation of serumBCAA is a molecular biomarker of the insulin-resistantstate (11–17). Moreover, Bcat2 knockout mice that areinsulin resistant have elevated circulating BCAAs (18). Inaddition, elevated serum BCAA in the setting of a high-fatdiet may activate the mammalian target of rapamycin-dependent serine phosphorylation of insulin receptor sub-strate-1 (19, 20). Although a variety of rodent and humanstudies have shown the branched-chain keto-acid dehy-drogenase complex plays a pivotal role in BCAA metab-olism, there is much still unknown about adipose tissueregulation of the BCAA pathway and the role of depot-specific functions (17). Herein we report a role of inflam-mation in controlling the BCAA/tricarboxylic acid (TCA)metabolism pathway, suggesting that the accumulation ofleucine, isoleucine, and valine in the serum is, at least inpart, a result of decreased BCAA and TCA cycle metab-olism selectively in inflamed visceral adipose tissue.

Research Design and Methods

AnimalsMale C57BL/6J mice were placed on a normal chow (�4%

fat by weight) or a high-fat (�35% fat by weight; Bio-ServF3282) diet at weaning (21). At 12–15 weeks of age, mice werekilled by cervical dislocation, and tissues were harvested, frozenin liquid nitrogen, and stored at �80°C until further processing.The University of Minnesota Institutional Animal Care and UseCommittee approved all experiments. The male thyroid re-sponse element-X-box binding protein 1 (Xbp1) and adipocyte-specific adiponectinP-rtTA transgenic mice (referred to as FIXsmice) were generated by the transgenic core facility at the Uni-versity of Texas Southwestern Medical Center and describedelsewhere (22, 23). Mice were maintained on a 12-hour dark,12-hour light cycle from 6:00 AM to 6:00 PM and housed ingroups of no more than five with unlimited access to water andchow (2916; Teklad) or doxycycline-containing (600 mg/kg)chow diets (Bio-Serv) and induced with doxycycline for 48hours.

Chemical reagentsRecombinant mouse TNF�, IL-6, IL-1�, and IFN� were pur-

chased from R&D Systems. Antibodies were obtained fromcommercial sources: �-actin from Sigma; and Bcat2, Sdha, Bck-dha, and Atp5a from Abcam. Isotopes of leucine were pur-

chased from PerkinElmer. Bay11–7085 was purchased fromCayman Chemical.

Quantitative RT-PCRExpression of mRNA was measured by quantitative RT-

PCR. Briefly, total RNA was isolated from tissue or 3T3-L1adipocytes using Trizol reagent (Invitrogen Corp) according tothe manufacturer’s protocol. RNA was treated with deoxyribo-nuclease I, and cDNA was synthesized using an iScript cDNAsynthesis kit (Bio-Rad Laboratories). Amplification was moni-tored with iQ SYBR Green Supermix and the MyiQ detectionsystem (BioRad Laboratories). Data were analyzed using the–��cycle threshold method and normalized to transcription fac-tor II element expression. Supplemental Table 1 contains thegene names, symbols, accession numbers, and primer sequencesused to amplify and detect the target transcripts.

Cell culture3T3-L1 fibroblasts were maintained and differentiated into

adipocytes as described previously (24). Differentiation was in-duced by the addition of DMEM containing 10% fetal bovineserum, 0.5 mM methylisobutylxanthine, 0.25 �M dexametha-sone, and 170 �M insulin. The methylisobutylxanthine anddexamethasone were removed after 2 days, and the insulin wasremoved after 4 days. The differentiated 3T3-L1 cells weremaintained in DMEM with 10% fetal bovine serum and used inexperiments 6–12 days after induction.

Leucine uptake and fractionationBriefly, day 8 differentiated adipocytes were treated with 1

nM TNF� for 24 hours, serum starved for 2 hours in DMEM,and radioactive leucine added for 5–180 minutes. Total cellularuptake was measured using [3H(3,4)]-leucine, whereas incorpo-ration into lipid and protein pools were measured using[14C(U)]-leucine. Fractionation of incorporated 14C-leucine wasaccomplished using a hexane-isopropanol-water (3:2:1) extrac-tion in which lipids portioned into the organic phase, whereasradioactive protein was collected into the precipitate.

Metabolite extraction and analysisPolar metabolites were extracted from cells or mouse serum

using a chloroform-methanol extraction procedure. Serum wasadded to a 1.5-mL centrifuge tube containing 500 �L chloro-form, 250 �L methanol, and 150 �L 0.9% sodium chloride. Themixture was vortexed and centrifuged at 13 000 � g for 10minutes. The upper aqueous/methanol layer was removed to anew tube, and the chloroform layer was washed with 1:1 0.9%sodium chloride-methanol. Three milliliters of methanol wereadded to the aqueous phase to facilitate drying under nitrogen.The dried samples were stored at �80°C until nuclear magneticresonance (NMR) analysis. Immediately prior to the NMR anal-ysis, the dried powder/film was resuspended in 100 �L 100 mMsodium phosphate in D2O (pH 7.4) containing 20 �M trimeth-ylsilyl propionic-2,2,3,3-day4 acid as a calibration standard.

NMR metabolite analysisNMR experiments were performed at 25°C on a Bruker

Avance III 700 MHz spectrometer with a 1.7 mm CryoProbe.One-dimensional noesy spectra were acquired with 128 tran-sients using 65 536 data points under fully relaxed conditions.

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Assignments were made using Chenomx version 7.5. Spectrawere exponentially line broadened by 1 Hz, Fourier trans-formed, and phase and baseline corrected. Spectra were cali-brated to an internal 20 �M trimethylsilyl-tetradeuterosodiumpropionate standard to establish metabolite concentrations andas a chemical shift reference. Metabolite assignments were madeaccording to the chemical shifts and pattern of coupling con-stants using the Chenomx compound library (Chenomx).

Immunoblot analysis3T3-L1 adipocytes treated with and without TNF� were

homogenized using a glass-teflon homogizer in homogenizationbuffer (20 mM Tris-HCl; 220 mM mannitol; 70 mM sucrose; 1mM EDTA; 0.1 mM EGTA, pH 7.4) supplemented with pro-tease inhibitors (Calbiochem). For Bcat2 analysis, homogenateswere centrifuged at 700 � g to remove nuclei, unbroken cells,and the lipid cake and mitochondria recovered by further cen-trifugation at 12 000 � g. Equal amounts of protein were sep-arated by SDS-PAGE, transferred to polyvinyl difluoride immo-bilon-FL (Millipore) membranes and blocked in Odysseyblocking buffer (LI-COR Biosciences). Membranes were incu-bated with infrared-conjugated secondary antibodies and visu-alized using Odyssey Infrared Imager (LI-COR Biosciences).

Statistical analysisAll values are expressed as mean � SEM. Statistical signifi-

cance was determined using the two-tailed Student t test assum-ing unequal variances, or where appropriate, a two-wayANOVA with Bonferroni post hoc analysis. Values of P � .05are considered significant (*) with an increased significance of avalue of P � .01 indicated (**).

Results

Obesity and inflammation down-regulateexpression of genes linked to BCAA metabolismselectively in adipose tissue and culturedadipocytes

Our laboratory has recently demonstrated that macro-phage-derived inflammatory factors down-regulate theexpression of genes linked to antioxidant biology in adi-pocytes and lead to mitochondrial dysfunction (25).Moreover, in mouse models, oxidative stress is stronglybiased toward the visceral relative to the subcutaneousdepot in which inflammation is the greatest, antioxidantgenes are down-regulated, reactive aldehydes accumu-late, and protein carbonylation is increased (26). Duringthe course of our studies, we also evaluated the expressionof genes linked to BCAA metabolism in adipose tissue ofhigh-fat-fed C57BL/6J mice and compared it with leanlittermates. Figure 1A shows a schematic representationof the proteins involved in BCAA uptake and mitochon-drial metabolism. Figure 1, B and C, show that relative tolean controls, the mRNA level of essentially every BCAA

metabolism pathway protein was down-regulated in theepididymal adipose tissue of high-fat-fed mice but not ininguinal fat. To assess whether the down-regulation of theBCAA pathway gene expression was adipose specific, apanel of enzymes was profiled in brown fat, muscle, andliver, and expression was found to not be affected and, inthe case of liver, was increased (Figure 1D). These resultsdemonstrate the pathway-specific down regulation of theBCAA metabolism enzymes in visceral adipose tissue.

Immune cell-derived inflammatory factors have beenshown to down-regulate many metabolic genes, and todetermine the effect of cytokines on BCAA metabolism,we treated 3T3-L1 adipocytes with TNF� and measuredthe mRNA levels of genes linked to BCAA metabolism.Figure 2A shows that essentially the entire BCAA path-way is coordinately down-regulated in response to TNF�.In addition, the expression of preadipocyte markers,branched-chain amino acid transaminase 1 and preadi-pocyte factor 1, were not increased by TNF� treatment,indicating that dedifferentiation is not occurring (Figure2A). We also observed a dose-dependent down-regula-tion with varying concentrations of TNF� (Figure 2B)and that other inflammatory factors (IL-6, IL-1�, andIFN�) similarly down-regulated BCAA gene expression(Figure 2C). Furthermore, the expression of branched-chain amino-acid transaminase 2 (Bcat2), the enzyme re-sponsible for the conversion of the BCAAs to their respec-tive �-ketoacids, and the branched chain keto-aciddehydrogenase-� subunit were also reduced similarly atthe protein level in response to TNF� (Figure 2D). Be-cause TNF� activates the canonical NF-�� pathway, weevaluated whether restriction of the NF-�B pathway byinhibiting I�B� phosphorylation with Bay11–7085would affect the expression of BCAA genes in response toTNF�. Figure 2E shows that for Slc1a5, Bcat2, and Bck-dha, treatment of day 8 3T3-L1 adipocytes with Bay11–7085 attenuated the TNF�-driven down-regulation, sug-gesting that NF-�� plays a primary role in the decreasedexpression of the BCAA genes.

Because BCAA metabolism serves as a source ofanaplerotic replenishment for the TCA cycle because leu-cine is metabolized to acetyl-CoA, isoleucine to acetyl-CoA, and succinyl-CoA and valine to succinyl-CoA, wecharacterized the mRNA expression of enzymes linked tothe TCA cycle enzymes in TNF� treated 3T3-L1 adi-pocytes and similarly found nearly the entire pathwaydown-regulated in response to cytokine treatment (Figure3A). Importantly, pyruvate carboxylase, the primarysource of anaplerotic oxaloacetate, was similarly down-regulated. Similarly, succinate dehydrogenase �-subunit(Sdha) protein levels were also decreased after TNF�

treatment (Figure 2D). Furthermore, we investigated the

doi: 10.1210/me.2014-1275 mend.endojournals.org 413


expression of the same panel of TCA enzymes in inguinal(Figure 3B) and epididymal (Figure 3C) white adiposetissue from chow and high-fat-fed mice and again foundepididymal fat to have reduced levels of expression,whereas inguinal fat remained largely unaffected, if notincreased. These results support the general considerationthat inflammation drives the expansion of the subcutane-ous depot while limiting the visceral depot through thetargeted regulation of the BCAA and TCA cyclepathways.

Transport of BCAAs and the metabolic effect ofenzyme down-regulation

To parallel the analysis of gene expression in the BCAApathway with metabolic processes, we evaluated BCAAtransport. Two systems facilitate BCAA uptake into adi-pocytes. Slc1a5 mediates direct BCAA transport of leu-cine, isoleucine, and valine, whereas Slc3a2 and Slc7a5form a heterodimeric plasma membrane complex ex-changing internal glutamine and asparagine for externalBCAA (27–30). As shown in Figure 4A, TNF� treatment

Figure 1. Obesity down-regulates the expression of genes of BCAA metabolism. Schematic diagram of cellular BCAA metabolism (A) andexpression of BCAA metabolism genes in epididymal (B) or inguinal white adipose tissue (C) in C57BL/6J mice maintained on a low-fat (filled bars)or high-fat (open bars) diet. D, Expression of BCAA metabolism genes in brown adipose tissue, liver, and gastrocnemius skeletal muscle tissue.Error bars represent SEM. *, P � .05 (n equals 6–12 per group).



of 3T3-L1 adipocytes down-regulated the expression ofSlc1a5 but did not affect the expression of either Slc3a2 orSlc7a5. Furthermore, expression of Slc1a5 was selectivelydown-regulated in the epididymal, but not subcutaneousdepot, of high-fat-fed mice (Figure 4B). To parallel thedown-regulation of Slc1a5 with transport, we evaluatedthe [3H]-leucine uptake in response to TNF� treatmentusing the 3T3-L1 adipocyte system. Figure 3C shows thatinflux was diminished approximately 50% in TNF�-treated adipocytes. To further investigate the fate of theinternalized leucine, we extracted the metabolites andmonitored the metabolism of [14-C]-leucine into proteinand lipid. Consistent with the down-regulation of the en-

tire BCAA pathway, the conversion of the leucine intotriglyceride was markedly attenuated (Figure 4D)whereas the conversion of the internalized leucine intoprotein was largely unaffected (Figure 4E).

Intracellular metabolomics of TNF�-treated 3T3-L1adipocytes

Because the metabolism of [14C]-leucine to lipid, butnot protein, was markedly attenuated, we analyzed thesteady-state level of intracellular metabolites as a mecha-nism toward understanding BCAA and TCA cycle metab-olism. As shown in Figure 5A, we measured an accumu-lation of intracellular BCAAs in TNF�-treated 3T3-L1s

Figure 2. Inflammatory factors down-regulate the expression of genes of BCAA metabolism. A, Expression of BCAA metabolism genes in 3T3-L1adipocytes treated on day 8 after differentiation for 24 hours with 1 nM TNF�. B, Expression of BCAA metabolism genes in 3T3-L1 adipocytestreated on day 8 after differentiation for 24 hours with varying concentrations of TNF�. C, Expression of BCAA metabolism genes in 3T3-L1adipocytes treated on day 8 after differentiation for 24 hours with 1 nM of the indicated cytokine. D, Expression of Bcat2, Sdha, Bckdha, Atp5a,and �-actin in 3T3-L1 adipocytes treated for 24 hours on day 8 after differentiation with 1 nM TNF�. E, Expression of BCAA metabolism genes in3T3-L1s treated on day 8 after differentiation for 24 hours with 1 nM TNF� and 10 �M Bay11–7085. $, P � .05 compared with TNF� treatment.ND, not detectable. Error bars represent SEM. *, P � .05 compared with control (n 6 per treatment group).



compared with controls. Furthermore, intracellular glu-tamine and asparagine levels were reduced after TNF�

treatment (Figure 5B), suggesting that the exchange ofglutamine/asparagine for BCAAs through the Slc7a5/Slc3a2 transport system may be compensating for re-duced transport through Slc1a5. Furthermore, a reduc-tion asparagine synthetase, which is observed in adiposetissue of ob/ob mice, could explain the reduction in as-paragine in TNF�-treated 3T3-L1 cells (31). In addition,we evaluated organic acid intermediates and measured anincrease of 2-oxoglutarate, a decrease in fumarate andpyruvate, and increased lactate levels (Figure 5C), consis-tent with decreased activity of the TCA cycle.

ER stress and the BCAA pathwayBecause inflammatory cytokines induce ER stress in

vivo and TNF� treatment of 3T3-L1 adipocytes similarlyinduces the unfolded protein response and activation of

Jnk (32, 33), we evaluated the effect of ER stress on theBCAA pathway. Two experimental systems were used toprobe the role of ER stress on the BCAA pathway. First,3T3-L1 cells were treated with either tunicamycin orthapsigargin to induce an accumulation of nonglycosy-lated and therefore unfolded proteins or inhibit the sar-coendoplasmic reticulum Ca2-ATPase, respectively. Asshown in Figure 6A, treatment with either tunicamycin orthapsigargin led to the down-regulation of genes linked tothe BCAA pathway. This result suggested that increasedER stress, or the ER stress response pathway, affects geneexpression of targets linked to mitochondrial BCAA me-tabolism. Extending these observations, tunicamycin orthapsigargin also down-regulated several key antioxidantgenes including Prdx3 and Gsta4 as well as the key mito-chondrial transcriptional coactivator peroxisomal prolif-erator-activated receptor-� coactivator 1� and mitochon-drial biogenesis signal regulator endothelial nitric oxidesynthase (data not shown). These results imply that ERstress reprograms mitochondrial systems broadly at thelevel of both mitochondrial biogenesis and metabolism.However, both tunicamycin and thapsigargin treatmentmay lead to secondary effects unrelated to ER stress, andas such we used a molecular genetic method specific to ERstress: the selective expression of spliced Xbp1 in adiposetissue.

Using transgenic mice expressing Xbp1s induciblydriven by the adiponectin promoter (referred to as FIXsmice), we evaluated subcutaneous and visceral adiposetissue from low-fat-fed FIXs and wild-type C57BL/6Jmice for the regulation of genes linked to the BCAA path-way. Because adiponectin is expressed to high levels insubcutaneous adipose tissue as well as visceral depot, weassessed the expression of spliced and total Xbp1 in thetwo locales. As shown in Figure 6B, Xbp1s was expressedin both depots but to the highest levels in the subcutane-ous site. Possibly with high level of Xbp1s mRNA in thesubcutaneous depot, the expression of genes linked to theBCAA pathway was markedly decreased in subcutaneousfat (Figure 6C) while only moderately decreased in thevisceral depot (Figure 6D). Similar to the results withtunicamycin and thapsigargin, the expression of key an-tioxidants Prdx3 and Gsta4 were markedly down-regu-lated in subcutaneous fat from the FIXs mouse as were themitochondrial biogenesis regulators endothelial nitric ox-ide synthase and peroxisomal proliferator-activated re-ceptor-� coactivator 1� (data not shown). These resultssuggest that even under conditions of low-fat feeding dur-ing which ER stress is not developed, transgenic expres-sion of Xbp1s drives the down-regulation of the BCAApathway. These results suggest that ER stress can lead tothe BCAA pathway down-regulation but does not dem-

Figure 3. Obesity and inflammation down-regulate TCA cycle andanaplerotic reaction enzyme gene expression. A, Expression of TCAcycle metabolism genes in 3T3-L1 adipocytes treated with or without 1nM TNF� for 24 hours. Expression of TCA cycle genes in inguinal whiteadipose tissue (B) and epididymal white adipose tissue (C) from low-fator high-fat fed C57BL/6J mice. Error bars represent SEM. *, P � .05(n 6–12 per group).



onstrate that ER stress is required for BCAA pathwaycontrol.

Discussion

The work described here profiles a previously unappreci-ated regulatory system controlling the branched-chainamino acid metabolism pathway and a potential expla-nation for the observation of elevated circulating BCAAsin insulin resistance and type 2 diabetes. It has been previ-ously shown that elevated circulating BCAAs are one of the

best predictive biomarkers for the development of type 2diabetes, but it has been unclear as to the mechanistic basisof this observation (9, 13, 14). Previous work from Hermanet al (9) has shown that reduced uptake of BCAA by adiposetissue may be sufficient to elevate circulating BCAA. How-ever, it is not likely that mitochondrial dysfunction per seleads to insulin resistance because in many cases, mitochon-drial dysfunction is compensated for by increased glycolysisand substrate-level phosphorylation that in turn reduces theserum glucose level (34).

To investigate the effect of inflammation on the BCAApathway, we first assessed the expression of BCAA me-

tabolism enzymes in adipose tissuefrom lean and obese mice. The ex-pression of BCAA genes in the epi-didymal depots of mice maintainedon high-fat diets were significantlydown-regulated relative to lean con-trol mice, whereas those in the ingui-nal depot were largely unaffected(Figure 1, B and C). The subcutane-ous depot exhibited a trend towardthe down-regulation of BCAA genesbut clearly not to the same extent asdoes visceral fat. Similarly, 3T3-L1adipocytes treated with the proin-flammatory cytokine, TNF�, anddemonstrated a coordinate down-regulation of essentially the entirepathway (Figure 2A), and the down-

Figure 4. Leucine transport and metabolism in 3T3-L1 adipocytes. A, Expression of leucine transport proteins in 3T3-L1 adipocytes treated for 24hours with 1 nM TNF�. B, Expression of leucine transport proteins in inguinal and epididymal white adipose tissue from mice maintained on chow(black bars) or high-fat diet (white bars) for 12 weeks. C, Total [3H]-leucine uptake in 3T3-L1 adipocytes treated with 1 nM TNF�. [14C]-leucinefractioned into lipid (D) or protein (E) after 24 hours TNF� treatment. Error bars represent SEM. *, P � .05 (n 6 per group).

Figure 5. Metabolomics analysis of 3T3-L1 cells. 3T3-L1 adipocytes were treated for 24 hourswith 1 nM TNF� and the intracellular levels of branched chain amino acids (A), amino acids (B),and organic acids (C) analyzed. Error bars represent SEM. *, P � .05 (n 6 per group).



regulation was measured in response to other inflamma-tory factors, suggesting that inflammation broadly affectsBCAA metabolism (Figure 2C). This effect was largelyablated when 3T3-L1s were cotreated with TNF� andBay11–7085, suggesting the observed down-regulation isat least in part dependent on the NF-�B pathway (Figure2E). Interestingly, IFN� that is made by T cells also down-regulated adipocyte BCAA metabolism genes, indicatingthat cells other than inflammatory macrophages may playa role in affecting adipocyte lipid metabolism (Figure 2C).Functional analysis of BCAA metabolism using [14C]-leu-cine incorporation into lipid pools corroborated the lossof BCAA metabolism with essentially no effect on aminoacid incorporation into protein (Figure 4, D and E). [3H]-leucine uptake is reduced approximately 50% due to thedown-regulation of Slc1a5, but even when the data arenormalized to uptake, conversion of leucine to lipid is dra-matically reduced (Figure 4C). Moreover, because increasedoxidative stress leads to potentiated carbonylation of BCAApathway enzymes (34), the mechanism of decreased BCAAmetabolism is likely a combination of both reduced expres-sion and carbonylation of pathway enzymes. Consistentwith reduced BCAA uptake but essentially no BCAA metab-olism, the intracellular pool of BCAA was increased inTNF�-treated adipocytes (Figure 5A).

The TNF�-mediated down-regulation of mitochon-drial metabolism was not restricted to the BCAA path-way. Indeed, our metabolomic analysis of TNF�-treated

3T3-L1 adipocytes also revealedchanges in the TCA cycle intermedi-ates (Figure 5C). Moreover, the ex-pression of several TCA cycle en-zymes was down-regulated at themRNA level after TNF� treatment(Figure 3A), and each of the TCAcycle enzymes is carbonylated underconditions of increased oxidativestress (34). The same pattern of TCAcycle enzymes being down-regulatedwas evident in visceral adipose tis-sue. It should also be stressed thatthe key enzyme controlling mito-chondrial oxaloacetate, pyruvatecarboxylase, is significantly down-regulated in epididymal fat andTNF� treated adipocytes (Figure 3Aand 3C). Because pyruvate carboxy-lase is a key lipogenic enzyme medi-ating the metabolism of glucose-de-rived pyruvate, the results are alsoconsistent with a decreased flux ofglucose into triglyceride in response

to inflammation. However, TCA cycle enzyme gene ex-pression is increased in subcutaneous adipose tissue (Fig-ure 3B), implying that inflammation biases nutrient up-take and metabolism away from the visceral depot andtoward the sc depot due to the attenuation of de novolipogenesis.

In many systems mitochondrial dysfunction is corre-lated with an increase in ER stress (35, 36). To determinewhether ER stress could influence mitochondrial dysfunc-tion, specifically BCAA metabolism, via regulation of theBCAA metabolism pathway, we used both pharmacolog-ical and molecular genetic methods to induce ER stress.Figure 6 shows that the pharmacological activation of ERstress either via inhbition of glycosylation (tunicamycin)or by inhibition of calcium reuptake (thapsigargin) re-sulted in a down-regulation of BCAA metabolism genes inthe 3T3-L1 cell culture system. Extending this analysis invivo, adipose-specific overexpression of spliced XBP1(FIXs) resulted in a down-regulation of BCAA geneslargely in subcutaneous adipose depots in which XBP1s isexpressed at the mRNA level to the greatest extent (Figure6B). Interestingly, such mice also exhibit increasedplasma leucine levels (Deng, Y., and P. E. Scherer, man-uscript in preparation), suggesting that ER stress is suffi-cient to cause adipose mitochondrial dysfunction as wellas decreased BCAA metabolism.

In summary, our results suggest that inflammation reg-ulates BCAA metabolism and lipogenesis in visceral adi-

Figure 6. ER stress down-regulates BCAA metabolism genes. A, 3T3-L1 adipocytes weretreated for 24 hours with 5 �M tunicamycin, 1 �M thapsigargin, or dimethylsulfoxide (DMSO),and the expression of BCAA/TCA genes were analyzed. B, Expression of spliced XBP1 inepididymal and subcutaneous fat of wild-type and FIXs mice. Expression of BCAA/TCAmetabolism genes in wild-type and transgenic mice overexpressing spliced XBP1 maintained on achow diet in the inguinal (C) or epididymal (D) depot are shown. Error bars represent SEM.*, P � .05 (n equals 4–8 per group).



pose tissue. Furthermore, the results presented suggestthat within the context of adult fat pad expansion, inflam-mation limits visceral adipose tissue while allowing forcontinued expansion of the subcutaneous depot via de-pot-specific regulation of mitochondrial metabolism.

Acknowledgments

We thank the members of the Bernlohr research group, whoprovided helpful suggestions regarding this work, as well as theMinnesota Supercomputing Institute for support in the dataanalysis. We also thank Todd Rappe and the Minnesota NMRCenter for assistance with the data collection and metabolomicanalyses. We also thank Drs Fredric Kraemer (Stanford Univer-sity) and Christopher Lynch (Penn State University) for helpfuldiscussions.

Address all correspondence and requests for reprints to: DrDavid A. Bernlohr, Department of Biochemistry, Molecular Bi-ology, and Biophysics, University of Minnesota-Twin Cities,Minneapolis, MN 55401. E-mail: [email protected].

This work was supported by National Institutes of HealthGrants R01 DK084669 (to D.A.B.), P01 DK088761 (to P.E.S.)and the Minnesota Obesity Center (National Institutes ofHealth Grant P30 DK050456).

Disclosure Summary: The authors have nothing to disclose.

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