R-α-Lipoic acid and acetyl-l-carnitine complementarily promote mitochondrial biogenesis in murine 3T3-L1 adipocytes

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1 Dietologi (28) 51: DOI 1.17/s ARTICLE R-α-Lipoic cid nd cetyl-l-crnitine complementrily promote mitochondril iogenesis in murine 3T3-L1 dipocytes W. Shen & K. Liu & C. Tin & L. Yng & X. Li & J. Ren & L. Pcker & C. W. Cotmn & J. Liu Received: 13 July 27 / Accepted: 13 Septemer 27 / Pulished online: 17 Novemer 27 # Springer-Verlg 27 Electronic supplementry mteril The online version of this rticle (doi:1.17/s ) contins supplementry mteril, which is ville to uthorised users. W. Shen : K. Liu : C. Tin : L. Yng : X. Li : J. Ren : L. Pcker Institute for Nutritionl Science, Shnghi Institutes for Biologicl Sciences, Chinese Acdemy of Sciences, Shnghi, Chin C. Tin : X. Li Grdute School of the Chinese Acdemy of Sciences, Beijing, Chin L. Pcker Deprtment of Moleculr Phrmcology nd Phrmceuticl Sciences, School of Phrmcy, University of Southern Cliforni, Los Angeles, CA, USA C. W. Cotmn : J. Liu () Institute for Brin Aging nd Dementi, University of Cliforni, 1261 Gillespie Neuroscience Reserch Fcility, Irvine, CA , USA e-mil: j.liu@uci.edu Astrct Aims/hypothesis The im of the study ws to ddress the importnce of mitochondril function in insulin resistnce nd type 2 dietes, nd lso to identify effective gents for meliorting insulin resistnce in type 2 dietes. We exmined the effect of two mitochondril nutrients, R-αlipoic cid (LA) nd cetyl-l-crnitine (ALC), s well s their comined effect, on mitochondril iogenesis in 3T3- L1 dipocytes. Methods Mitochondril mss nd oxygen consumption were determined in 3T3-L1 dipocytes cultured in the presence of LA nd/or ALC for 24 h. Mitochondril DNA nd mrna from peroxisome prolifertor-ctivted receptor gmm nd lph (Pprg nd Ppr) nd crnitine plmitoyl trnsferse 1 (Cpt1), s well s severl trnscription fctors involved in mitochondril iogenesis, were evluted y rel-time PCR or electrophoretic moility shift (EMSA) ssy. Mitochondril complexes proteins were mesured y western lot nd ftty cid oxidtion ws mesured y quntifying CO 2 production from [1-14 C] plmitte. Results Tretments with the comintion of LA nd ALC t concentrtions of.1, 1 nd 1 μmol/l for 24 h significntly incresed mitochondril mss, expression of mitochondril DNA, mitochondril complexes, oxygen consumption nd ftty cid oxidtion in 3T3L1 dipocytes. These chnges were ccompnied y n increse in expression of Pprg, Ppr nd Cpt1 mrna, s well s incresed expression of peroxisome prolifertor-ctivted receptor (PPAR) gmm coctivtor 1 lph (Pprgc1), mitochondril trnscription fctor A (Tfm) nd nucler respirtory fctors 1 nd 2 (Nrf1 nd Nrf2). However, the tretments with LA or ALC lone t the sme concentrtions showed little effect on mitochondril function nd iogenesis. Conclusions/interprettion We conclude tht the comintion of LA nd ALC my ct s PPARG/A dul lignds to complementrily promote mitochondril synthesis nd dipocyte metolism. Keywords Mitochondril complex. Mitochondril trnscription fctor A. Nucler respirtory fctor 1. Nucler respirtory fctor 2. Peroxisome prolifertorctivted receptor gmm. Peroxisome prolifertor-ctivted receptor lph. Peroxisome prolifertor-ctivted receptor. Gmm coctivtor 1 lph Arevitions ALC cetyl-l-crnitine EMSA electrophoretic moility shift

2 166 Dietologi (28) 51: KRH LA mtdna PPAR Introduction Kres Ringer solution uffered with HEPES R-α-lipoic cid mitochondril DNA peroxisome prolifertor-ctivted receptor Mitochondril dysfunction plys centrl role in wide rnge of ge-ssocited disorders nd vrious forms of cncer [1]. Mitochondril glucose nd ftty cid metolism in muscle nd dipocytes is impired in ptients with insulin resistnce nd type 2 dietes [2], while mitochondril loss in dipose tissue is correlted with the development of type 2 dietes [3]. Peroxisome prolifertor-ctivted receptors (PPARs) re lignd-ctivted trnscription fctors elonging to the nucler receptor superfmily [4]. Ech PPAR memer displys tissue-selective expression pttern nd hs distinct roles in lipid metolism. Pprg, which is required for dipose tissue formtion, hs emerged s trnscriptionl regultor of metolism nd plys n importnt role in dietes nd oesity [5]. Ppr is centrlly involved in mitochondril iogenesis nd ftty cid oxidtion [6]. Incresed production of Pprgc1, key regultor of mitochondril iogenesis, my e involved in oesity nd the pre-dietic stte in skeletl muscle [7]. Therefore, regulting Pprg/ ctivity nd dipocyte metolism my improve insulin sensitivity nd glucose disposl [2, 5, 8]. Promoting mitochondril iogenesis y upregultion of the Pprgc1 pthwy hs een suggested s strtegy for preventing nd reversing insulin resistnce, oesity nd dietes [3, 9, 1]. In this regrd, severl drugs hve een tested, such s metformin nd 5-minoimidzole-4-croxmide rionucleoside [11], the Pprg gonist pioglitzone/rosiglitzone nd Ppr gonist WY-14,643 [12 14], s well s et 3-drenergic receptors gonist CL-316,243 [15] nd oestrogen-relted receptor α [16]. Dietry supplementtion with micronutrients my complement phrmcologicl gents in the prevention nd tretment of dietes, with prticulr focus on the prevention of dietic complictions [17]. R-α-Lipoic cid (LA) nd cetyl-l-crnitine (ALC) hve een found to e protective nutrients of mitochondri [18]. LA is n ntioxidnt nd exogenously supplied LA cn e NADHnd NADPH-dependently reduced in mitochondri nd cytosol [19]. LA hs een shown to ct s n NADH oxidse inhiitor to lock oxidnt production nd decrese phgocytosis of myelin y mcrophges [2]. LA is involved in mitochondril α-keto cid dehydrogense complexes tht ctlyse oth crohydrte nd mino cid metolism. LA hs lso een found to e le to stimulte glucose uptke y ctivting the insulin signlling pthwy in dipose nd muscle cells [21]. However, LA lso inhiits differentition of 3T3-L1 pre-dipocytes induced y hormonl mixture or troglitzone [22] nd cuses n increse in oxidnts t reltively high concentrtions (5 1, μmol/l) in 3T3-L1 dipocytes [23]. LA hs een reported to function s wek dul PPARG/A [24] nd to promote weight loss, meliorte insulin resistnce nd therogenic dyslipidemi, s well s to lower lood pressure. ALC is the cetyl derivtive of L-crnitine, which is required for the trnsport of long-chin ftty cids into the mitochondri for β-oxidtion, ATP production nd for the removl of excess short- nd medium-chin ftty cids [25]. LA nd/or ALC improve mitochondril function in geing rts nd their comintion ppers more potent owing to complementry effects [25 28]. LA [29] nd ALC [3, 31] hve een tested in severl lrge clinicl trils on prevention or tretment of dietes nd its complictions. Both nutrients result in n improvement in insulin sensitivity, nd evidence suggests eneficil effects on crdiovsculr prmeters ssocited with the metolic syndrome nd type 2 dietes. The present study sought to determine whether tretment of dipocytes with LA nd/or ALC ffects mitochondril mss nd the expression of genes nd proteins involved in mitochondril iogenesis. Methods Mterils Anti-β-ctin ws from Sigm (St Louis, MO, USA), nti-oxphos complex I, II, III nd IV from Invitrogen (Crlsd, CA, USA), the reverse trnscription system kit from Promeg (Mnnheim, Germny) nd HotStrTq from Tkr (Otsu, Shig, Jpn). Nrf1, Nrf2, Pprgc1, 18S rrna nd β-ctin primers were synthesised y Biosi Biotech (Shnghi, Chin). ALC (hydrochloride slt) ws from Sigm Tu (Pomezi, Itly) nd LA (tris slt) ws gift from K. Wessel, Vitris, Bd Homurg, Germny. TRIzol nd other regents for cell culture were from Invitrogen. Cell culture nd differentition 3T3-L1 cells hve een extensively used s model of dipogenic differentition nd insulin ction. 3T3-L1 cells undergo growth rrest nd initite progrmme of differentition mnifested y lrge lipid droplet ccumultion upon hormonl stimultion. In prllel, these cells ecome sensitive to insulin, express Glut4 nd disply insulin-induced ctivtion of glucose uptke comprle to tht seen in primry dipose cells [12]. In the present study, murine 3T3-L1 pre-dipocytes (Americn Type Culture Collection, Mnsss, VA, USA) were cultured in DMEM supplemented with 1% (v/v) fetl ovine serum nd llowed to rech confluence. Differentition of pre-dipocytes ws initited with 1. μmol/l insulin,

3 Dietologi (28) 51: μmol/l dexmethsone nd.5 mmol/l 3-isoutyl-1- methylxnthine in DMEM supplemented with 1% (v/v) fetl ovine serum. After 48 h, the culture medium ws replced with DMEM supplemented with 1% fetl ovine serum nd 1. μmol/l insulin. The culture medium ws chnged every other dy with DMEM contining 1% (v/v) fetl ovine serum. Cells were used t 9 to 1 dys following differentition induction when exhiiting 9% dipocyte phenotype. Mitochondril mss A fluorescent proe (Mito-Trcker Green FM; Moleculr Proes, Eugene, OR, USA) ws used to determine the mitochondril mss of dipocytes, i.e. more ccurtely: the frctionl volume of tht prt of n dipocyte tht is occupied y mitochondri. Adipocytes treted with LA nd/or ALC for 24 h were trypsinised nd centrifuged t 3, g t 4 C for 5 min, resuspended in Kres Ringer solution uffered with HEPES (KRH) nd.1% BSA (w/v) nd then incuted with.1 μmol/l MitoTrcker Green FM in KRH uffer for 3 min t 37 C. Cells were centrifuged t 3, g t 4 C for 5 min nd resuspended in 4 μl fresh KRH uffer. Fluorescence ws nlysed y FACS Cliur (Becton Dickinson, Mountin View, CA, USA). Electron microscopy 3T3-L1 dipocytes t dy 8 of differentition were seeded on glss coverslips. On dy 9, cells were treted with LA (1 μmol/l) nd/or ALC (1 μmol/l) for 24 h. On dy 1, dipocytes were fixed overnight with 2.5% (v/v) glutrldehyde in.1 mol/l sodium phosphte uffer (ph 7.3). They were postfixed with 2% (w/v) OsO 4 in the sme uffer, followed y lock stining with 1% (w/v) urnyl cette. After dehydrtion with grded series of ethnol, they were wshed y propylene oxide nd emedded in Spurr s low viscosity resin. Silver to gold sections were cut nd exmined using trnsmission electron microscope (CM 1; Philips, Eindhoven, the Netherlnds) t 6 kv ccelerting voltge [32]. Mesurements were mde on ten individul dipocytes treted with or without LA nd/or ALC. For ech individul dipocyte profile in the re, the numer of mitochondri nd the totl mitochondril section re were determined. All electron microscopic photogrphs were nlysed lind with regrd to tretments. Mitochondril DNA Totl DNA ws extrcted using kit (QIAmp DNA Mini kit; Qigen, Hilden, Germny) nd quntittive PCR ws done using 18S rrna primers for nucler trget sequence nd primers for mitochondril DNA trget using mitochondril D-loop. Quntittive PCR ws performed using rel-time PCR system (Mx3P; Strtgene, Amsterdm, the Netherlnds). Rections were performed with 12.5 μl SYBR-Green Mster Mix (ABI, Wrrington, UK),.5 μl of ech primer (1 μmol/l) nd 1 ng templte (DNA) or no templte (NTC), with RNsefree wter eing dded to finl volume of 25 μl. The cycling conditions were s follows: 5 C for 2 min, initil denturtion t 95 C for 1 min, followed y 4 cycles of 95 C for 3 s, 55 C for 1 min nd 72 C for 3 s. Ech quntittive PCR ws performed in triplicte. The following primers were used: mitochondril D-loop forwrd, 5 - AATCTACCATCCTCCGTG-3, reverse 5 -GACTAAT GATTCTTCACCGT; 18S rrna forwrd: 5 -CATTC GAACGTCTGCCCTATC-3 nd reverse: 5 -CCTGCT GCCTTCCTTGGA-3. The mouse 18S rrna gene served s the endogenous reference gene. The melting curve ws done to ensure specific mplifiction. The stndrd curve method ws used for reltive quntifiction. The rtio of mitochondril D-loop to 18S rrna ws then clculted. Finl results re presented s percentge of control. Cell respirtion Oxygen consumption y intct cells ws mesured s n indiction of mitochondril respirtion ctivity. We used the BD Oxygen Biosensor System (BD Biosciences, Sn Diego, CA, USA), n oxygen-sensitive fluorescent compound (Tris 1,7-diphenyl-1,1 phennthroline ruthenium [II] chloride) emedded in gs-permele nd hydrophoic mtrix permnently ttched to the ottom of multiwell plte. The concentrtion of oxygen in the vicinity of the dye is in equilirium with tht in the liquid medium. Oxygen quenches the dye in concentrtiondependent mnner. The fluorescence correltes directly to oxygen consumption in the well. The unique technology llows homogeneous instntneous detection of oxygen levels. After tretment, dipocytes were wshed in KRH uffer plus.1% (w/v) BSA. Cells from ech condition were divided into liquots in triplicte in BD Oxygen Biosensor System plte (BD Biosciences). The numer of cells contined in equl volumes ws not sttisticlly significnt in response to vrious nutrient tretments nd concentrtions. Pltes were seled nd red on fluorescence spectrometer (Moleculr Devices, Sunnyvle, CA, USA) t 1 min intervls for 6 min t n excittion of 485 nm nd emission of 63 nm [13]. Results re expressed s the slope of fluorescence intensity. RNA isoltion nd reverse trnscription-polymerse chin rection After incution, cells were wshed twice with ice-cold PBS. Totl RNA ws isolted using the single-step TRI regent nd 1 μg RNA ws reverse-trnscried into cdna. In rief, the isolted RNA ws dissolved in sterile wter nd 2.5 mmol/l Mg 2+, 1 mmol/l dntps,.5 μg oligodt 15, 25 U AMV reverse trnscriptse, 1 RT uffer, giving finl volume of 2 μl. The smple ws incuted t 25 C (1 min), 42 C (6 min) nd 99 C (5 min). cdna ws diluted in DNse-free wter (1:25) efore quntifiction y rel-time PCR.

4 168 Dietologi (28) 51: The primers for quntifiction of mrna y rel-time quntittive PCR for Nrf1, Nrf2, Tfm, Ppr, Pprg, Pprgc1, Cpt1 nd β-ctin mrnas re listed in the Electronic supplementry mterils (ESM Tle 1). Quntittive PCR ws performed using Mx3P (see ove). Ech quntittive PCR ws performed in triplicte. The mouse β-ctin gene served s the endogenous reference gene. The evlution of reltive differences of PCR product mong the tretment groups ws crried out using the ΔΔCT method. The reciprocl of 2CT (used CT s n exponent for the se 2) for ech trget gene ws normlised to tht for β-ctin, followed y comprison with the reltive vlue in control cells. Finl results re presented s percentge of control. Western lot nlysis After tretment with either or oth LA nd ALC, cells were wshed twice with ice-cold PBS, lysed in smple uffer [62.5 mmol/l Tris Cl ph 6.8, 2% (w/v) SDS, 5 mmol/l dithiothreitol (DTT)] t room temperture nd vortexed. Cell lystes were then oiled for 5 min nd clered y centrifugtion (13, g, 1 min t 4 C). Protein concentrtion ws determined using protein ssy (Bio-Rd DC; Hercules, CA, USA). The solule lystes (1 μg per lne) were sujected to 1% (w/v) SDS-PAGE; proteins were then trnsferred to nitrocellulose memrnes nd locked with 5% (w/v) non-ft milk/tris-uffered sline Tween 2 (TBST) for 1 h t room temperture. Memrnes were incuted overnight t 4 C with primry ntiodies directed ginst β-ctin (1:5,), nti-oxphos Complex I (NADH uiquinol oxidoreductse 39 kd suunit, 1:2,), nti-oxphos Complex II (succinteuiquinone oxidoreductse 7 kd suunit, 1:2) nd nti-oxphos Complex III (uiquinol cytochrome c oxidoreductse core II, 1:2,) in 5% (w/v) milk/tbst. After wshing memrnes with TBST three times, memrnes were incuted with horserdish peroxidse-conjugted secondry ntiody for 1 h t room temperture. Western lots were developed using electrochemoluminescence (Roche, Mnnheim, Germny) nd quntified y scnning densitometry [33]. Fig. 1 Increse of mitochondril mss in dipocytes treted with LA nd/or ALC. Adipocytes were stimulted with LA nd/or ALC from.1 to 1 μmol/l for 24 h nd mitochondril mss ws estimted y MitoTrcker (1 nmol/l) stining using flow cytometry. The rightwrd shift in the curve represents n increse in mitochondril mss. The curves were generted using Becton Dickinson FACS Cliur using Cell Quest softwre. The curves were modified with smooth function of the softwre. M1 nd M2 indicte the oundries etween cells tht were negtive (M1) or positive (M2) for Mito-Trcker Green FM; these were ritrrily set on cells without stin (not shown) nd with stin for Mito- Trcker Green FM smples. LA nd/or ALC.1 μmol/l; LA nd/or ALC 1. μmol/l; c LA nd/or ALC 1 μmol/l; d LA nd/or ALC 1 μmol/l. Green, LA tretment; red, ALC tretment; lue, LA+ALC tretment. e 3T3-L1 dipocytes were incuted with LA nd/or ALC t the indicted concentrtions. Results re presented s percentge of untreted control cells. Vlues re men±sem of the results from four independent experiments. p<.5 nd p<.1 vs control Counts Counts Reltive fluorescence intensity Counts LA (µmol/l) ALC (µmol/l) Counts c d e

5 Dietologi (28) 51: Mitochondril isoltion Following ddition of trypsin, the cells were pelleted y centrifugtion t 3 g for 5 min t 4 C. All of the susequent steps were performed on ice. The resulting pellet ws then resuspended in.5 ml of mitochondril isoltion uffer (215 mmol/l mnnitol, 75 mmol/l sucrose,.1% BSA, 1 mmol/l EGTA, 2 mmol/l HEPES, ph 7.2) nd homogenised on ice with 2 ml glss homogeniser (Dounce, Fisher Scientific, Pittsurgh, PA, USA). The mitochondri were then purified y differentil centrifugtion t 1,3 g for 5 min to pellet unroken cells nd the nuclei. The superntnt frction ws then centrifuged t 13, g for 1 min to pellet the mitochondri. The pellet ws resuspended in EGTA-free isoltion uffer [34]. Electrophoretic moility shift ssy Binding ctivity of mitochondril trnscription fctor A (Tfm) ws ssessed y electrophoretic moility shift ssy (EMSA) ccording to Knzw et l. [34]. Briefly, rdioctive proe contining the nucleotide sequence of the hevy-strnd promoter of Tfm ws prepred y nneling pired oligonucleotides with the sequences 5 -TTTCCTCCTAAC TAAACCCTCTTTAC-3 nd 5 -GTAGGCAAGTAAA GAGGGTTTAGTTA-3 nd ws lelled using γ- 32 P- lelled ATP ( Bq/mmol; Amershm Biosciences, Buckinghmshire, UK) nd T4 polynucleotide kinse (Promeg, Mnnheim, Germny). The protein DNA inding protein rection ws performed t room temperture for 2 min in volume of 2 μl. The rection mixture contined 1 μg mitochondril protein, 1 μg/ml poly di dc, 1 mmol/l Tris HCl (ph 7.5), 5 mmol/l NCl,.5 mmol/l EDTA,.5 mmol/l dithiothreitol, 1 mmol/l MgCl 2, 4% glycerol (v/v) nd 1, cpm-lelled nucleotides. Protein DNA complexes were resolved y electrophoresis on 6% (w/v) crylmide gel nd sujected to utordiogrphy. For competition ssys, nonlelled oligonucleotides were dded t 5-fold molr excess to the rection mixture efore the ddition of the mitochondril protein extrct. Ftty cid oxidtion Following incution, 14 CO 2 ws mesured ccording to the method of Thupri et l. [35] with some modifictions. Adipocytes were preincuted for 3 min with 1.5 ml of the following uffer: 114 mmol/l NCl, 4.7 mmol/l KCl, 1.2 mmol/l KH 2 PO 4, 1.2 mmol/l MgSO 4, 11 mmol/l glucose. After preincution, 2 μl of ssy uffer ws dded contining 114 mmol/l NCl, 4.7 mmol/l KCl, 1.2 mmol/l KH 2 PO 4, 1.2 mmol/l MgSO 4, 11 mmol/l glucose nd 2.5 mmol/l plmitte (contining.37 MBq [1-14 C] plmitte) ound to lumin, nd the cells were incuted t 37 C for 2 h. After incution, the plte ws clmped nd seled, nd perchloric cid ws injected into the medium through the holes in the lid, driving CO 2 through the tunnel into n djcent well, where it ws trpped in 1 mol/l NOH. After trpping, liquots of NOH nd medium were trnsferred into scintilltion vils nd rdioctivity ws mesured on multipurpose scintilltion counter (LS 65; Beckmn Coulter, Fullerton, CA, USA). Cells were collected into.3 ml.5% (w/v) SDS for susequent protein mesurement. All ssys were performed in duplicte nd dt were normlised to protein content. Blnks were prepred y dding 5 μl 6%(w/v) perchloric cid to the cells efore incution with the ssy uffer for 2 h. Sttisticl nlysis All dt re representtive of t lest three independent experiments. Dt re presented s mens±se. Sttisticl significnce ws determined y using one-wy ANOVA with Bonferroni s post hoc tests etween the two groups. The criterion for significnce ws set t p<.5. c e Surfce re (µm 2 ) nd density (no. of mitochondri/1 µm 2 of cytoplsmic re) d Are Density Fig. 2 Electron microscopy of LA nd/or ALC-treted 3T3-L1 dipocytes. d Mitochondril morphometry in dipocytes (mgnifiction 1,). Illustrtions of whole-cell profiles, with mitochondril profiles surrounding lipid droplet indicted y rrows. On dy 8 of differentition, 3T3-L1 dipocytes were seeded on to coverslips. On dy 9, cells ( d) were treted for 24 h with LA 1 μmol/l, ALC 1 μmol/l nd LA 1 μmol/l+alc 1 μmol/l or left untreted (). e Morphometric nlysis of surfce re of the mitochondri nd mitochondril density ws performed. Vlues re mens±sem. p<.5 vs control. White rs, control; grey rs, LA; striped rs, ALC; lck rs, LA+ALC

6 17 Dietologi (28) 51: Fig. 3 Effect of LA nd/or ALC on levels of mitochondril protein nd DNA. 3T3-L1 dipocytes were treted for 24 h with LA nd/or ALC. Cells were susequently soluilised into SDS smple uffer nd nlysed y western lotting with ntiodies ginst β-ctin nd mitochondril electron trnsport complex I, II nd III. Immunolots re representtive of stedy-stte levels of proteins. Quntittive nlyses of the nds for mitochondril complex I, II nd III y densitometry. Results re presented s percentge of untreted control cells. Vlues re men±sem from four independent experiments. p<.5 vs control. White rs, control; grey rs, LA; striped rs, ALC; lck rs, LA+ALC. c 3T3-L1 dipocytes were treted for 24 h with LA nd/or ALC. PCR products were quntified for fluorescence using SYBR Green. Quntittive vlues were tulted for D-loop: 18S rrna rtio. Results re presented s percentge of untreted control cells. Dt re men±sem (n=5). p<.5 vs control tken s 1; p<.1 vs control tken s 1. White rs, LA; grey rs, ALC; lck rs, LA+ALC Complex I Complex II Complex III β -ctin c LA (μmol/l) ALC (μmol/l) Reltive protein level MtDNA (D-loop/18S rrna) rtio Complex I Complex II Complex III (μmol/l) (μmol/l) Results LA nd ALC increse dipocyte mitochondril mss To determine whether LA nd ALC led to modifiction of mitochondril mss, the dye, MitoTrcker Green FM, which ccumultes in mitochondri, ws used to lel nd quntify mitochondri in dipocytes. Seven dys fter differentition ws initited, 3T3-L1 dipocytes were exposed to LA nd/or ALC for 24 h. Tretment with either LA or ALC t.1 to 1 μmol/l resulted in trend towrds dose-dependent increse in fluorescence intensity, ut these increses were not significnt. However, the comintion of LA nd ALC sttisticlly significntly incresed the reltive fluorescence intensity in the concentrtion rnge of.1 to 1 μmol/l, these increses eing higher thn those found with LA or ALC lone (Fig. 1). Electron microscopic nlysis of dipocyte mitochondri LA (1 μmol/l) nd/or ALC (1 μmol/l) tretment ltered the size of individul mitochondri nd their structure (Fig. 2). A quntittive nlysis (ten cells nlysed) showed tht ALC (Fig. 2c) did not ffect mitochondril re nd numer, while LA (Fig. 2) showed trend towrds incresing mitochondril re nd numer, lthough this ws not sttisticlly significnt. However, the comintion of LA nd ALC tretment (Fig. 2d) cused significnt increse in the totl mitochondril section re (p<.5) nd lso n increse (187.8±24%) in the verge numer of mitochondril profiles per cell, counted in doule-linded fshion in five imges contining whole-cell profiles sectioned through the middle of the nucleus. Production of OxPhos complex I, II, III nd IV proteins nd mitochondril DNA Becuse LA nd/or ALC tretment

7 Dietologi (28) 51: Fig. 4 Oxygen consumption in 3T3-L1 dipocytes. Equl volumes of cells were seprted into liquots in wells of 96- well BD Oxygen Biosensor plte. Pltes were covered nd fluorescence in ech well ws recorded over time with fluorescence microplte spectrophotometer. Representtive oxygen consumption curves. Quntittive chnges in the respirtory rte of dipocytes during ech condition were clculted y determining the kinetic mesurements. V mx is the mximum oxygen consumption rte. Vlues re men±sem; results re presented s percentge of untreted control cells from three independent experiments. p<.5 vs controls tken s 1; p<.1 vs control tken s 1. White rs, LA; grey rs, ALC; lck rs, LA+ALC O 2 consumption (fluorescence intensity 1 5 ) V mx Time (s) (μmol/l) LA+ALC (1 μmol/l) ALC (1 μmol/l) LA (1 μmol/l) Control ltered the size of individul mitochondri nd their pprent numer, western lotting ws used to estimte the ctul increse in mitochondril complexes cused y LA nd/or ALC tretment. LA showed n increse in mitochondril OxPhos Complex III protein t 1 μmol/l (33±1.2%). The comintion tretment of equl concentrtions of LA nd ALC showed significnt increse (p<.5 vs control) in the levels of OxPhos Complex I t.1+.1 μmol/l (279± 21.8%), OxPhos Complex II t μmol/l (19±13.9%) nd OxPhos Complex III t oth.1+.1 μmol/l (329± 16.1%) nd μmol/l (31±.23%; Fig. 3). LA nd/ or ALC showed no effect on the expression of mitochondril OxPhos complex IV in dipocytes (dt not shown). As D-loop is known to e the mjor site of trnscription initition on oth the hevy nd light strnds of mitochondril DNA (mtdna), we exmined in vitro whether LA nd/or ALC could increse mtdna expression. As shown in Fig. 3c, the comintion of LA nd ALC incresed the rtio of mitochondril D-loop/18S rrna in the concentrtion rnge of.1 to 1 μmol/l, with ll increses sttisticlly significntly higher thn those found with either LA or ALC lone. Oxygen consumption To determine whether incresed mitochondril iogenesis is ccompnied y chnges in oxygen consumption, cells were treted with LA nd/or ALC t.1 to 1 μmol/l. As shown in Fig. 4, the sl rte of oxygen consumption ws sttisticlly significntly incresed in dipocytes treted with the comintion of LA nd ALC in the concentrtion rnge of.1 to 1 μmol/l, ll increses eing significntly higher thn those found with either LA or ALC lone. Expression of mitochondril iogenesis genes Pprgc1 is coctivtor tht promotes mitochondril iogenesis nd mitochondril ftty cid oxidtion. The reltive undnce of mrna trnscripts encoding for Pprgc1, Nrf1 nd Nrf2 were exmined y quntittive RT-PCR. Tretment of LA t.1 to 1 μmol/l resulted in trend towrds dosedependent increse in expression of trnscripts encoding for Pprgc1, ut ALC did not ffect expression of trnscripts encoding for Pprgc1 nd Nrf1 nd Nrf2. However, the comintion of LA nd ALC incresed undnce of trnscripts encoding for Pprgc1 nd Nrf1 nd Nrf2 significntly in the concentrtion rnge of.1 to 1 μmol/l; ll increses were sttisticlly significntly higher thn those found with either LA or ALC lone (Fig. 5 c). The trnscription fctor Tfm is involved in regulting expression of nucler genes encoding mjor mitochondril proteins tht regulte mtdna trnscription nd repliction. In the ESMA ssy, competition rection ws performed y preincuting 5-fold molr excess of unlelled oligonucleotide representing the Tfm inding site with the isolted mitochondri. The specific of Tfm inding of ctivity ws confirmed y the sence of Tfm complex nd in the negtive control (mutnt Tfm proe). A dosedependent increse in Tfm inding ws found with LA nd ALC, respectively; the comintions of LA nd ALC lso showed significnt stimultion t concentrtions of.1 to 1 μmol/l (Fig. 5d). mrna of Pprg, Ppr, Cpt1 nd ftty cid oxidtion The reltive undnce of mrna trnscripts encoding for Pprg, Ppr nd Cpt1 were exmined y quntittive RT-PCR. Tretment of LA t 1 μmol/l resulted in increse

8 172 Dietologi (28) 51: c Reltive Pprgc1 mrna Reltive Nrf1 mrna Reltive Nrf2 mrna (μmol/l) (μmol/l) (μmol/l).5) t 1 μmol/l, ut LA hd no effect t the sme concentrtion. However, the comintion of LA nd ALC significntly incresed ftty cid oxidtion y 323% (p<.1). Discussion White dipose tissue is n importnt endocrine orgn involved in the control of whole-ody metolism nd insulin sensitivity. Thus, mitochondril iogenesis could in prt underlie the centrl role of dipose tissue in the control of whole-ody metolism nd the ctions of some insulin sensitisers [12]. Indeed, it hs een reported tht mitochondril dysfunction might e n importnt contriuting fctor in insulin resistnce nd type 2 dietes [2], while mitochondril loss in dipose tissue is correlted with the development of type 2 dietes [3]. Hence it is possile tht stimultion of mitochondril iogenesis my reduce the effects of mitochondril loss of function. The comintion of reltively low doses of LA nd ALC improved mitochondril function nd my provide possile therpeutic intervention for preventing nd treting insulin resistnce nd type 2 dietes. d Tfm LA (μmol/l) ALC (μmol/l) Fig. 5 Effect of tretment with LA nd/or ALC on expression of Pprgc1, Nrf1, Nrf2 nd Tfm mrna in dipocytes. Adipocytes were incuted with LA nd/or ALC for 24 h nd totl RNA ws isolted. Pprgc1, Nrf1 nd Nrf2 mrna ws nlysed y quntittive RT-PCR with gene-specific oligonucleotide proes in dipocytes. Effect of LA nd/or ALC on expression of Pprgc1; effect of LA nd/or ALC on expression of on Nrf1; c effect of LA nd/or ALC on expression of on Nrf2. The cycle numer t which the vrious trnscripts were detectle ws compred with tht of β-ctin s n internl control; results re expressed s percentge of untreted control cells. All vlues re men±sem of four independent experiments. p<.5 vs controls tken s 1; p<.1 vs controls tken s 1. White rs, LA; grey rs, ALC; lck rs, LA+ALC. d Anlysis of the inding ctivity of mitochondril proteins to n Tfm proe. Autordiogrph of EMSA performed with 32 P-lelled mutnt Tfm nucleotide nd mitochondril protein extrct isolted from LA nd/or ALC t the indicted concentrtions in expression of Pprg, Ppr nd Cpt1. ALC lso incresed the expression of Ppr nd Cpt1 significntly t 1 μmol/l, ut showed no effect on Pprg. However, the comintion of LA nd ALC sttisticlly significntly incresed the undnce of Ppr nd Cpt1 t concentrtion of 1 μmol/l, ll increses eing significntly higher thn those found with LA or ALC lone (Fig. 6). As shown in Fig. 6, ALC incresed ftty cid oxidtion y 223% (p< Reltive mrna Plmitte oxidtion Control LA ALC LA+ALC Fig. 6 Effect of LA nd/or ALC on expression of Pprg, Ppr nd Cpt1 mrna, nd on ftty cid oxidtion in dipocytes. Adipocytes were incuted with LA nd/or ALC t 1 μmol/l for 24 h. Pprg, Pprα nd Cpt1 mrna were nlysed y quntittive RT-PCR with gene-specific oligonucleotide proes of dipocytes. The cycle numer t which the vrious trnscripts were detectle ws compred with tht of β-ctin s n internl control nd results expressed s percentge of untreted control cells. All vlues re men±sem of four independent experiments. p<.5 vs controls tken s 1; p<.1 vs controls tken s 1. White rs, LA; grey rs, ALC; lck rs, LA+ALC Adipocytes were incuted with LA nd/or ALC for 24 h nd [ 14 -C] plmitte oxidtion ws mesured. Vlues re men±sem; results re presented s percentge of untreted control cells from three independent experiments. p<.5 vs controls; p<.1 vs controls Pprg Ppr Cpt1

9 Dietologi (28) 51: Pprg plys n importnt role not only in dipogenesis, ut lso in regulting lipid metolism in mture dipocytes [12]. PPARG ctivity cn e modulted y direct inding of low moleculr weight lignds, some of which re cliniclly effective glucose-lowering gents, leit with dverse side effects tht limit their utility [5]. Activtion of Pprg y glucose-lowering gents such s thizolidinedione, highffinity gonist lignd for Pprg, led to net flux of ftty cids from the circultion nd other tissues into dipocytes [5]. Interestingly, incresed ft storge did not increse the size of dipocytes, ut rther led to smller dipocytes, possily due to incresed dipocyte differentition nd ctivtion of Pprgc1, which promotes mitochondril iogenesis. Ppr is lso known to e n importnt regultor of mitochondril iogenesis nd β-oxidtion in tissues like hert nd liver [4, 36]. As shown in our experiments, Ppr nd Pprg levels were upregulted y LA nd ALC tretment in 3T3L1 dipocytes. This upregultion closely correltes with the stimultion of mitochondril iogenesis nd induction of CPT1 involved in ftty cid oxidtion, suggesting these nutrients my ct s PPARG/A lignds to increse ftty cid uptke, increse dipocyte differentition, nd ctivte Pprgc1 to promote mitochondril iogenesis in 3T3L1 dipocytes. Since LA nd ALC re nutrients without pprent side effects, they might exert etter PPARg/ lignd stimultion thn glucose-lowering gents or other lignds. The Pprg gonist pioglitzone nd Ppr gonist WY-14,643 were le to increse Pprgc1 expression nd mtdna copy numer, s well s enhncing the oxidtive cpcity of white dipose tissue leding to insulin sensitistion [12, 13, 37]. Mitochondril iogenesis nd remodelling in white dipocyte tissue enhnces ftty cid uptke nd oxidtion y incresed oxygen consumption. Consistent with the morphologicl dt, oxygen consumption in dipocytes ws incresed when dipocytes were treted with LA nd ALC, indicting tht dipocytes treted with comintion of LA nd ALC hve greter mitochondril mss thn cells treted with LA or ALC lone. In vivo, n increse in fttycid oxidtion my protect ginst dipocyte hypertrophy under conditions where incresed uptke of ftty cids occurs from the circultion. Thus, the effect of LA nd ALC my contriute directly nd indirectly to chnges in whole-ody energy metolism nd insulin sensitivity. The mechnisms of the protective effects of the comintion of LA nd ALC re not cler, ut might include [18, 38, 39]: (1) protection of mitochondri from oxidtive dmge nd thus slowing down of the loss of mitochondri; (2) stimultion of repir of less dmged mitochondri; (3) stimultion of degrdtion of more dmged mitochondri (lysosomes); nd (4) stimultion of de novo mitochondril iogenesis. The complementry effect of LA nd ALC on cognitive nd mitochondril dysfunction hs een shown in geing rts [27, 28, 4]. One reson is tht LA+ALC ct on different pthwys necessry for mitochondri: LA is mitochondril ntioxidnt nd cofctor of pyruvte dehydrogense, while ALC is n energy enhncer [25, 41]. Another possiility is tht ALC, lthough stimulting mitochondril function, my cuse side effects of oxidtive stress in mitochondri [42], while LA, n effective mitochondril ntioxidnt, is le to meliorte tht side effect of ALC. The complementry effect my lso come from the different functions of LA nd ALC on the four vrious spects. In conclusion, the strong synergistic effect of the comintion of LA nd ALC in 3T3L1 dipocytes suggests tht these two nutrients complement ech other s function in mitochondril iogenesis. 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