Kev Noj Qab Haus Huv Cocoa Flavanols Txhim Kho Mitochondrial Function hauv Skeletal Muscle Thiab Hloov Kho Tag Nrho Lub Cev Metabolism hauv Cov nas noj qab haus huv

Yog xav paub ntxiv, hu rau:tina.xiang@wecistanche.com

Abstract: Mitochondrial dysfunction yog dav tshaj tawm nyob rau hauv ntau yam kab mob thiab pab txhawb rau lawv pathogenesis. Peb soj ntsuam cov nyhuv ntawmcocoa flavanolssupplementation ntawm mitochondrial muaj nuj nqi thiab tag nrho cov metabolism, thiab peb tshawb xyuas seb lub mitochondrial deacetylase sirtuin -3 (Sirt3) puas koom los yog tsis. Peb tshawb nrhiav qhov cuam tshuam ntawm 15 hnub ntawm CF supplementation hauv cov tsiaj qus thiab Sirt3-/ nas. Tag nrho lub cev metabolism tau raug soj ntsuam los ntawm kev tsis ncaj calorimetry, thiab kev ntsuas qhov ncauj ntawm qhov ncauj tau ua los ntsuas.qabzib metabolism. Mitochondrial ua pa ua haujlwm tau raug soj ntsuam hauv cov fibers permeabilized thiab cov ntsiab lus ntawm pyridine nucleotides (NAD ntxiv thiab NADH) tau txheeb xyuas. Hauv hom tsiaj qus, CF supplementation tau hloov pauv tag nrho lub cev metabolism los ntawm kev txhawb nqa kev siv carbohydrate thiab txhim kho cov qabzib siab ntev. CF supplementation induced ib tug tseem ceeb nce ntawm mitochondrial loj, thaum tseem ceeb qualitative adaptation tshwm sim los tuav H2O2 ntau lawm thiab cellular oxidative kev nyuaj siab. CF supplementation induced ib tug tseem ceeb nce nyob rau hauv NAD ntxiv thiab NADH cov ntsiab lus. Tag nrho cov teebmeem uas tau hais los saum toj no tau hais tsis tseeb hauv Sirt3-7 nas. Sib sau ua ke, CF supplementation boosted NAD metabolism uas nkoos sirtuins metabolism thiab txhim kho mitochondrial muaj nuj nqi, uas yuav pab tau rau cov kev soj ntsuam tag nrho lub cev metabolism adaptation, muaj peev xwm ntau dua los siv carbohydrates, tsawg kawg yog ib feem ntawm Sirt3.

Ntsiab lus: cocoa flavanols; NAD metabolism; mitochondrial loj; cov metabolism hauv qabzib; cev nqaij daim tawv

nyem kom tau txais xov xwm ntxiv

1. Taw qhia

Mitochondria yog subcellular organelles uas koom nrog ntau lub zog ntawm tes, xws li kev hloov hluav taws xob los ntawm mitochondrial oxidative phosphorylation thiab hauv mitochondrial hydrogen peroxide ntau lawm lossis mitochondrial-mediated cell tuag ua kom [1-3]. Cov ntaub ntawv pov thawj tshwm sim qhia tau hais tias mitochondrial tsis ua haujlwm tau koom nrog ntau yam pathologies thiab ua lub luag haujlwm tseem ceeb hauv lawv cov kab mob [4]. Peb pab pawg tau tshaj tawm txog kev puas tsuaj mitochondrial hauv cov neeg mob uas muaj ntshav qab zib hom 1 ntev ua ntej kev kho mob nyuaj [5]. Ntxiv mus, qhov txo qis ntawm mitochondrial oxidative muaj peev xwm tshwm sim nrog kev laus txawm nyob rau hauv cov neeg noj qab haus huv [6]. Yog li, kev txheeb xyuas cov tswv yim los txo cov kev ua haujlwm tsis zoo lossis txhim kho mitochondrial huab hwm coj yog lub sijhawm los tiv thaiv, lossis tsawg kawg kom txo qis, qhov tshwm sim ntawm ntau yam teeb meem lossis ua kom muaj peev xwm aerobic ntawm cov neeg noj qab haus huv.

Nyob rau hauv cov ntsiab lus no, kev txheeb xyuas ntawm kev nyab xeeb thiab ntuj tebchaw uas txhim kho mitochondrial muaj nuj nqi thiab tag nrho lub cev metabolism nrog los yog tsis txwv kev phiv yog txaus siab. Ntawm cov natural compounds,cocoa flavanols(CF) yog suav tias yog cov khoom cog lus zoo li lawv qhov kev noj qab haus huv tau pom tias muaj txiaj ntsig zoo rau kev noj qab haus huv ntawm lub plawv, insulin tsis kam, lossis kev tiv thaiv kab mob [7]. Qhov tseeb, CF tau pom tias txhim kho cov piam thaj metabolism, thiab kev tswj hwm luv luv ntawm CF yog ua raws li kev nce hauv insulin rhiab heev, txawm nyob rau hauv cov neeg mob ntshav qab zib tsis txaus siab [8,9].

Ntawm cov flavanols, (-)-epicatechin (EPI) yog qhov feem ntau pom monomer hauv CF, thiab EPI yog suav tias yog bioavailable thiab bioactive molecule ntawm CF [10]. Ib lub cev zoo ib yam ntawm cov pov thawj qhia tau hais tias EPI supplementation txhim kho mitochondrial muaj nuj nqi thiab / lossis cov ntsiab lus hauv cov leeg pob txha [11]. EPI supplementation txhawb ntau txoj hauv kev sib koom ua ke ntawm peroxisome proliferator-activated receptor gamma coactivator 1-alpha(PGCl) thiab loj nuclear transcriptional complexes. EPI ncaj qha txhim kho nitric oxide tiam, uas txhawb nqa mitochondrial biogenesis [11]. Concomitantly, cov sirtuins 1 thiab 3 kuj txhawb nqa los ntawm EPI supplementation [11]. Kev siv cov proanthocyanidins, oligomericflavonoidsuas muaj EPI, tau pom tias yuav ua rau kom intracellular nicotinamide adenine dinucleotide (NAD ntxiv) qib los ntawm kev nce ntawm ntau lub precursors rau NADbiosynthesis thiab upregulate sirtuin-1 mRNA qib hauv nas siab [12]. Tsev neeg sirtuin (Sirt), NAD-dependent deacetylase, muaj xya tus tswv cuab uas txawv los ntawm lawv cov subcellular faib, substrate specifications, thiab cellular functions [13]. Sirt1, ib qho kev kawm dav dav ntawm tsev neeg no, txhawb nqa mitochondrial biogenesis los ntawm kev txhawb nqa deacetylation ntawm PGCl, yog li txhim kho nws cov haujlwm transcriptional [14]. Sirte, qhov tseem ceeb metabolic sensor, tau txhawb nqa tom qab EPI kev tswj hwm [12,15-17]. Txawm li cas los xij, kev cuam tshuam ntawm EPI ntawm lwm cov sirtuins tsis tau tshawb pom. Qhov kev ntsuam xyuas ntawm EPI cov nyhuv yuav yog qhov tshwj xeeb tshaj yog nthuav rau sirtuins nyob rau hauv lub mitochondria (Sirt3, Sirt 4, thiab Sirt5), uas paub los hloov cov kev ua ntawm Krebs voj voog thiab ua pa saw enzymes [18]. Sirt3, uas tau qhia ntau heev hauv cov ntaub so ntswg nrog cov metabolism hauv siab thiab cov ntsiab lus mitochondrial, yog qhov txaus siab txog nws lub luag haujlwm tseem ceeb hauv kev tswj hwm lub cev mitochondrial los ntawm kev thim rov qab cov protein lysine deacetylation [19-21] Ntxiv mus, Sirt3 plays lub luag haujlwm tseem ceeb hauv Kev tswj hwm ntawm tag nrho lub cev metabolism [20. Sirt3 tau pom tias tau hloov pauv hauv cov leeg pob txha ntawm cov qauv ntawm hom 1 thiab hom 2 ntshav qab zib thiab cov kab mob plawv [22,23]. Tsis tas li ntawd, nws tau pom tias kev ua kom Sirt3 tuaj yeem sawv cev rau qhov kev cog lus kho mob rau kev txhim kho mitochondrial muaj nuj nqi thiab cov metabolism [23,24]. Yog li, txiav txim siab seb Sirt3 underlies ib feem ntawm cov txiaj ntsig ntawm CF supplementation ntawm tag nrho lub cev metabolism thiab kev ua haujlwm mitochondrial yog qhov txaus siab.

Hauv txoj kev tshawb no, peb tau siv txoj hauv kev sib koom ua ke los tshawb xyuas, hauv cov nas, cov txiaj ntsig ntawm 15-hnub CF ntxiv. Peb xav tias CF supplementation yuav: (i) hloov tag nrho lub cev metabolism thiabqabzib metabolism, (ii) nce mitochondrial muaj nuj nqi nyob rau hauv oxidative thiab glycolytic nqaij nyob rau hauv permeabilized fibers, thiab (ii) boost NAD metabolism. Peb kuj tau tshawb xyuas qhov kev koom tes ntawm Sirt3 hauv CF-induced mitochondrial biogenesis thiab tag nrho lub cev metabolism. Peb cov kev xav tau yog tias CF qhov cuam tshuam rau tag nrho lub cev metabolism thiab cov mitochondrial loj yuav blunted hauv Sirt3-7 nas.

2. Cov txheej txheem

2.1. Tsiaj thiab Noj Zaub Mov

Rau txoj kev tshawb no, 129S1/SvlmJ 10-cov nas muaj hnub nyoog ib lim piam tau yuav los ntawm Jackson Laboratories (Bar Harbor, ME, USA). Txhawm rau txiav txim siab seb sirtuins puas koom nrog CF kev tswj hwm-induced mitochondrial huab hwm coj thiab ua haujlwm hloov pauv, peb kuj tau siv 10-lub lim tiam Sirt3 tag nrho-lub cev tshem tawm cov txiv neej nas ntawm tib hom tsiaj qus [25]. Sirt3 / nas tau siv los tshawb nrhiav kev koom tes ntawm Sirt3 hauv CF-induced mitochondrial biogenesis thiab tag nrho lub cev metabolism hloov pauv. Cov nas tau khaws cia rau hauv ib chav ntawm qhov ntsuas kub ntawm 23-25 degree thiab tswj teeb pom kev zoo (12-h lub teeb thiab lub voj voog tsaus). Cov tsiaj tau nkag mus rau nas chow thiab kais dej ad libitum.

Tom qab tsawg kawg ib lub lim tiam ntawm kev nyob hauv cov chaw tsiaj, cov tsiaj sim tau txais CF supplementation los ntawm qhov ncauj gavage ntawm ib tug natural extract (302.1 mg / kg lub cev hnyav ob zaug ib hnub rau 15 hnub) resuspended nyob rau hauv ib tug carboxymethylcellulose (Sigma-Aldrich, St. , Mo, USA). Cov muaj pes tsawg leeg ntawm cov hmoov cocoa tau piav qhia hauv Table 1 thiab tau txais los ntawm Naturex (Quart de Poblet, Spain). Cov muaj pes tsawg leeg tau tsim los ntawm kev ua haujlwm siab ua kua chromatography txoj kev thiab 100 mg ntawm cov extract yog sib npaug rau qhov nruab nrab ntawm 475 mg ntawm qhuav cocoas noob. Cov nas tswj tau txais lub tsheb uas muaj cov ntsiab lus zoo sib xws ntawm theobromine thiab caffeine yaj hauv carboxymethylcellulose los ntawm qhov ncauj gavage. Cov koob tshuaj niaj hnub tau txiav txim siab raws li kev lag luam cov lus qhia uas hloov cov tib neeg koob tshuaj rau tsiaj sib npaug raws li thaj chaw ntawm lub cev, thiab peb muab cov tib neeg koob tshuaj los ntawm 12.3 [26]. Qhov ncauj gavage tau ua los ntawm tus kws tshaj lij.

2.2. Tissue Collection 1

Nees nkaum plaub teev tom qab qhov kawg ntawm qhov ncauj gavage, cov tsiaj tsis yoo mov tau muab tshuaj loog siv ketamine thiab xylazine (feem ntau: 100 thiab 10 mg / kg). Kev sau cov ntaub so ntswg tau ua tam sim ntawd tom qab ua tiav cov tshuaj loog. Lub soleus thiab dawb feem ntawm gastrocnemius raug tshem tawm. Cov qauv tau khov tam sim ntawd hauv cov kua nitrogen thiab khaws cia ntawm -80 degree rau kev soj ntsuam tom qab lossis siv los npaj cov nqaij tawv nqaij.

2.3. Kev ntsuam xyuas metabolic thiab lub cev ua haujlwm

Indirect calorimetry thiab spontaneous lub cev ua si tau raug ntsuas los ntawm kev siv lub Comprehensive Laboratory Animal Monitoring System (CLAMS; Columbus Instruments, Columbus, OH, USA). Cov nas tau nyob ib leeg hauv cov chav ntawm 28 degree, nrog lub teeb ntawm 07h00 txog 19h00 thiab ad libitum nkag mus rau nas chow thiab kais dej. Cov nas tau nkag mus rau cov kab mob metabolic rau peb hnub ua ntej hnub ib ntawm kev sau cov ntaub ntawv. Kev sau cov ntaub ntawv tau ua nyob rau hnub kaum tsib ntawm kev ntxiv. Cov pa oxygen noj (VO2), carbon dioxide ntau lawm (VCO2), thiab kev sib pauv ua pa (RER) tau ntsuas nrog cov cua ntws ntawm 0.5 L / min thiab cov qauv huab cua ntawm 0.4 L / min. Feem pua ​​​​ntawm cov txheeb ze sib txuas zaus (PCRF) ntawm RER tau txiav txim siab nyob rau lub sijhawm 24-h raws li tau piav qhia los ntawm Riachi li al. (2004). Luv luv, PCRF tau txiav txim siab los ntawm qhov sib ntxiv ntawm qhov ntau zaus ntawm txhua cov ntaub ntawv taw qhia mus rau qhov nce qib dhau los (los ntawm 0.65 txog 1.20 nrog qhov nce ntawm 0.01) [27]. Cov metabolic yooj tau txiav txim siab los ntawm Hill txoj kab nqes (H tus nqi) thiab 50 feem pua ​​(EC50) ntawm PRCF nkhaus thiab los ntawm kev ntsuas RER amplitude tshaj 24 h (Riachi li al.2004). Kev ua ub no ntawm lub cev tau saib xyuas siv lub tshuab hluav taws xob infrared uas tau kuaj pom qhov ua x- thiab z-axis. Ob qho tag nrho suav (txhua lub sijhawm ntawm lub nqaj tawg) thiab kev suav cov tsheb thauj neeg mob (txhua lub sijhawm cov nqaj tshiab tawg) raug soj ntsuam. X-tag nrho (X-TOT) suav tau sau npe thaum tus tsiaj txav kab rov tav ntau dua 0.5 hauv thiab yog tus sawv cev ntawm cov haujlwm me me, rov ua dua, xws li khawb thiab tu tu. X-ambulatory suav (X-AMB) ntsuas qhov kev txav chaw tiag tiag los ntawm kev sau npe suav tsuas yog thaum tus tsiaj tsoo lub nqaj tshiab. Z-tag nrho (Z-TOT) suav tau sau npe thaum 1.5 hauv kev txav ntsug tshwm sim, piv txwv li, kev txhawb nqa (Abreu-Vieira 2016). Rau kev kwv yees carbohydrate thiab rogoxidationlos ntawm VO2 thiab VCO2, peb tau siv cov lus tsis muaj protein ntau ua pa los ntawm Péronnet thiab Massicotte yav dhau los siv rau hauv nas [28,29].

2.4. Tag Nrho Lub Cev thiab Fat Mass

Tag nrho cov rog thiab tag nrho cov lean loj tau txiav txim siab nyob rau hauv cov nas uas tsis tau tshuaj xyuas los ntawm kev txheeb xyuas qhov kev cuam tshuam ntawm kev siv EchoMRI tag nrho lub cev sib nqus resonance analyzer (Echo Medical System, Houston, TX, USA). Cov rog tag nrho sawv cev rau tag nrho cov rog hauv lub cev. Tag nrho cov lean loj suav nrog cov leeg nqaij thiab sab hauv. Cov leeg nqaij pob txha yog paub los suav rau qhov loj tshaj plaws ntawm tag nrho cov lean loj.

2.5.Oral Glucose Tolerance Test (OGTT)

Glycemia tau ntsuas los ntawm kev siv cov ntshav poob los ntawm tus Tsov tus tw nrog glucometer (Accu-Chek Performa, Roche, Argentina). OGTT tau ua rau cov nas yoo mov rau 5 txog 6 teev. Qhov ntsuas tau ua nyob rau hnub kawg ntawm CF supplementation. Tom qab ntsuas qhov basal glycemia (lub sij hawm O), cov nas tau txhaj tshuaj rau ib os nrog kev daws ntawm 20 feem pua ​​​​qabzib hauv cov dej tsis huv ntawm koob tshuaj 1.5 g glucose·kg lub cev hnyav-1. Glycemia tau ntsuas ntawm 15, 30, 60,90, thiab 120 feeb tom qab txhaj tshuaj ntawm cov piam thaj. Tom qab ntawd, qhov nce ntxiv ntawm cov piam thaj nkhaus tau suav ua qhov ntsuas ntawm cov piam thaj siab.

2.6.qRT-PCR Kev Ntsuam Xyuas

The forward (F) and reverse (R) primers of the selected genes used in this study are presented in Table 2. Total RNA was obtained from pulverized frozen white gastrocnemius muscle using Trizol reagent (Invitrogen Life Technologies, Rockville, MD, USA). The purity, integrity, and quantity of RNA were evaluated using a microplate reader (Synergy H1(Biotek Instruments, Winooski, Vermont). The sample was considered pure when the ratio OD260/OD280 was>1.8. cDNA tau tsim los ntawm tag nrho RNA (2 μL) siv iScript rov qab hloov pauv supermix rau RT-qPCR (Biorad, Hercules, CA, USA). Tag nrho cov khoom RT tau soj ntsuam los ntawm ntau cov tshuaj tiv thaiv polymerase saw (PCR) siv 2x Platinium SYBR Green qPCR SuperMix-UDG raws li cov chaw tsim khoom tshwj xeeb (Invitrogen Lige Technologies, Burlington, Canada). Cycling tau ua tiav hauv CFX96 cycler (Biorad, Hercules, California; cov xwm txheej: 95 degree C rau 10 min thiab 40 cycles ntawm 95 degree rau 30 s, 55 degree rau 45 s thiab 72 degree rau 45 s). Thaum kawg ntawm txhua qhov kev khiav, qhov tsis muaj qhov tsim ntawm primer-dimer thiab muaj qhov tshwj xeeb amplicon tau lees paub tias siv qhov nkhaus melting. Lub voj voog lub voj voog (Ct) qhov tseem ceeb tau siv ua raws li 2-A△Ct txoj kev los tshuaj xyuas qPCR cov ntaub ntawv, nrog rau xws liß-Actin lossis HPRT1 ua vaj tse nyob 【30】. Quantitative RT-PCR qhov tseem ceeb tau cuam tshuam nrog cov pab pawg tswj hwm, uas tau teeb tsa rau 1.

2.7. Kev Ua Haujlwm Enzyme

Kev ntsuas ntawm cov haujlwm tshwj xeeb ntawm electron thauj saw complexes thiab citrate synthase tau ua spectrophotometrically raws li tau piav qhia yav dhau los [31]. Pulverized khov dawb gastrocnemius nqaij (~ 30 mg) tau homogenized nrog vibrating microbead homogenizer nyob rau hauv 500 uL ntawm homogenization tsis (120 mM KCl, 20 mM ntawm HEPES, 2mM ntawm MgCl2, 1 mM ntawm EGTA, thiab 5 mg·mL-I ntawm BSA , pH7.4) ua raws li qhov sib ntxiv ntawm 500 μL ntawm hypotonic media (25 mM potassium phosphate thiab 5 mM MgCl, pH 7.2). Cov qauv no tau muab xa mus rau peb lub voj voog ntawm lub voj voog khov-thaw hauv cov kua nitrogen. Cov qauv no tau centrifuged rau 10 min ntawm 600 × g ntawm 4 degree, thiab cov supernatant tau khaws cia rau hauv dej khov kom txog thaum kev soj ntsuam. Cov ntsiab lus protein tau txiav txim siab hauv cov supernatant hauv triplicate siv Pierce BCA Protein Assay Kit (Thermo Scientific, Rockford, IL, USA).

Cov kev ua ntawm citrate synthase (CS) tau txiav txim siab spectrophotometrically ntawm 412 nm tom qab txo qis ntawm 2 mM 5,5'-dithio-bis (2-nitrobenzoic acid) nyob rau hauv muaj 0.1 mM acetyl -CoA thiab 12 mM ntawm oxaloacetic acid hauv 200 mM Tris tsis kam (pH 7.4). rotenone-sensitive NADH-decylubiquinone oxidoreductase (complex I) assay tau ua nyob rau ntawm 340 nm siv tus txais 2, 3-dimethoxy-5-methyl-6-n-decyl{22}}benzoquinone ( 80 μM) thiab NADH li electron pub (200 μM) hauv 10 mM Tris tsis (pH 8.0). Qhov sib ntxiv ntawm 4μM ntawm rotenone tau siv los ntsuas qhov kev ua haujlwm rotenone-rhiab heev. Kev ua haujlwm ntawm cytochrome oxidase (complex IV) tau ua ntawm 550 nm siv 10μM txo cytochrome c ua tus pub thiab 2.5 mM ntawm n-dodecyl-ß-maltoside kom permeabilize ob lub mitochondrial membranes hauv 100 mM potassium phosphate 7.0p ().

Txhua qhov kev ntsuam xyuas enzyme tau txiav txim siab hauv triplicate, thiab cov txiaj ntsig tau cuam tshuam nrog cov pab pawg tswj hwm, uas tau teem rau 100.

2.8. Kev ntsuam xyuas Mitochondrial

Mitochondrial respiration thiab mitochondrial H2O2 ntau lawm tau kawm nyob rau hauv situ nyob rau hauv saponin permeabilized fibers siv dawb gastrocnemius thiab soleus nqaij|1]. Luv luv, fibers raug cais nyob rau hauv lub binocular microscope nyob rau hauv cov tshuaj A ntawm 4 degree (nyob rau hauv mM:2.77 CaK2 EGTA,7.23 K, EGTA,6.56 MgCl2, 20 taurines,0.5 DTT,5{ {50}} K-methane sulfonates, 20 imidazoles, 5.7 Na2 ATP thiab 15 creatine-phosphate, pH 7.1) thiab permeabilized hauv cov tshuaj A nrog 50 ug·mL-Iof saponin rau 30 min ntawm 4 degree. Mitochondrial ua pa ua haujlwm tau txiav txim siab hauv oximeter nruab nrog Clark-hom electrode (Oxygraph, Hansatech Instruments, Glasgow). Lub chamber tau ntim nrog 1 mL ntawm cov tshuaj B (hauv mM: 2.77 CaK, EGTA, 7.23 K, EGTA, 6.56 MgCl,, 20 taurine, 0.5 DTT, 50 K-methane sulfonates, thiab 20 imidazoles, pH 7.1) thiab tom qab kaw. Cov ntsiab lus ntawm cov pa oxygen hauv lub chamber, ib pob ntawm 1-2 mg qhuav qhov hnyav ntawm permeabilized myofibres tau muab tso rau hauv lub chamber, uas tau muab kaw. Tom qab kev nyeem ntawv hauv paus, cov hauv qab no ntxiv tau ua ntu zus: palmitoyl carnitine thiab malate (160 μM∶5 mM), glutamate (10 mM), succinate (25 mM), rotenone (0.5 μm), uncoupler CCCP (1 μM), -A (8 uM), thiab N, N', N'-tetramethyl-p-phenylenediamine dihydrochloride thiab ascorbate (0.9: 9 mM). Ua pa tau ntsuas ntawm 23 degree nyob rau hauv kev nplawm tas li. Thaum kawg ntawm txhua qhov kev sim, fibers tau ua tib zoo tshem tawm ntawm lub xov tooj xylographic, blotted, thiab qhuav yam tsawg 24 teev ntawm ~ 80C rau kev txiav txim siab ntawm fiber ntau. Tus nqi ntawm O2 noj (JO2) tau qhia hauv nmol O2 min-1.(mg qhuav hnyav)-I. Tag nrho cov kev ntsuas tau ua yam tsawg kawg hauv qhov sib npaug.

Net H2O2 tso tawm los ntawm respiring mitochondria yog ntsuas nyob rau hauv permeabilized fiber bundles nrog lub fluorescent sojntsuam Amplex Liab (20 μM∶ excitation-emission∶563-587 nm), raws li tau piav yav tas los [32]. Tom qab kev npaj ntawm permeabilized fibers, kuaj

destined rau H2O2 ntsuas tau rinsed peb zaug nyob rau hauv tsis Z (hauv mM:110 K-Mes, 35 KCl,1 EGTA,5 K2HPO4, 3MgCl,6H2O, thiab 0.5 mg:mL{ {14}} BSA, pH7.3 ntawm 4 degree). Fiber bundles ({{20}}}.3-1.0 mg qhuav hnyav) tau incubated ntawm 37 degree nyob rau hauv ib tug quartz microcuvette nrog nruam sib nqus stirring nyob rau hauv 600 uL ntawm tsis Z (pH 7.3 ntawm 37C) ntxiv nrog 1.2 UmL-I horseradish peroxidase. Lub hauv paus fluorescence nyeem tau raug coj mus rau qhov tsis muaj ib qho exogenous ua pa substrates. Cov tshuaj ntxiv hauv qab no tau ua ntu zus: glutamate (5 mM), succinate (5 mM), rotenone (0.5 uM), ADP (10 mM), thiab antimycin-A (8 μM). ua tib zoo tshem tawm ntawm lub cuvette, blotted, thiab qhuav los txiav txim siab fiber ntau. Tus nqi ntawm H2O2 ntau lawm tau qhia nyob rau hauv AU·min-1.(mg qhuav hnyav)-I thiab ib lub peev xwm oxphos uas sib haum mus rau lub siab tshaj plaws mitochondrial ua pa nyob rau hauv lub xeev phosphorylation. Tag nrho cov kev ntsuas tau ua yam tsawg kawg hauv qhov sib npaug.

Mitochondrial calcium retention capacity (CRC) tau soj ntsuam hauv cov nqaij dawb gastrocnemius dab fibers raws li tau piav qhia yav dhau los [33]. Luv luv, fibers tau incubated nyob rau hauv ib tug quartz microcuvette nyob rau hauv tas li stirring nyob rau hauv 600 μL ntawm CRC tsis (hauv mM∶250 sucrose, 10 MOPS, 0.005 EGTA, thiab 10P;-Tris, pH7.3). Fibers ces raug rau ib leeg mem tes ntawm 20 nM ntawm Ca2 ntxiv. Cov kev hloov pauv hauv extramitochondrial calcium concentration tau soj ntsuam fluorometrically siv Calcium ntsuab 5N (1 mM: excitation-emission:505-535} nm). Permeability transition pore susceptibility (PTP) tau soj ntsuam los ntawm kev ntsuas lub sij hawm xav tau rau PTP qhib, thiab CRC raug coj raws li tag nrho cov nyiaj ntawm Ca 'sau los ntawm mitochondria ua ntej nws tso tawm. Thaum kawg ntawm txhua qhov kev sim, fibers tau ua tib zoo tshem tawm ntawm lub cuvette, blotted, thiab qhuav los txiav txim siab fiber ntau. CRC qhov tseem ceeb tau qhia hauv nM ntawm Ca2 ntxiv rau ib mg ntawm qhov hnyav qhuav.

2.9. Western Blot Analysis

Proteins ntawm dawb gastrocnemius thiab soleus cov leeg tau diluted hauv Laemmli tsis thiab sib cais ntawm Mini-PROTEAN TGX Stain-Free 10 feem pua ​​precast polyacrylamide gels (Biorad); tus qauv sab hauv tau thauj khoom ntawm txhua gel. Kev sib cais electrophoretic tau ua tiav ntawm 200 V rau 35 feeb hauv kev tsiv teb tsaws chaw (25 mM TrisBase, 0.2 M glycine, thiab 1 feem pua ​​SDS (p/v)). Stain-Free(SF) thev naus laus zis muaj cov khoom muaj txiaj ntsig tri halo uas cuam tshuam nrog cov proteins, ua rau lawv kuaj pom los ntawm UV raug. SF imaging tau ua tiav siv ChemiDoc MP Imager thiab Image Lab4.0.1 software (Biorad, Hercules, California, USA) nrog rau 5-min stain activation time, thiab tag nrho cov qauv protein tau pom yog li ntawd. Cov proteins tau raug xa mus rau ntawm daim ntawv 0.2 um nitrocellulose siv Trans-Blot Turbo Transfer System (Biorad). Qhov zoo ntawm kev hloov pauv tau tswj los ntawm cov duab thaij duab siv SF thev naus laus zis. Tom qab cov kauj ruam hloov mus, cov carbonyl proteins tau derivatized nrog dinitrophenylhydrazine diluting hauv 2N HCL thiab thaum kawg tau ntxuav nrog methanol. Cov daim nyias nyias tau thaiv nrog 5 feem pua ​​​​ntawm cov mis tsis muaj rog hauv Tris-buffered saline uas muaj Tween-20 (TBST: 15 mM Tris / HCl, pH7.6,140 mM NaCl, thiab 0.05 feem pua ​​Tween-20) rau 1 h ntawm chav tsev kub. Membranes ces incubated ntawm 4 degree thaum hmo ntuj los yog 2 h nyob rau hauv chav tsev kub nrog cov thawj cov tshuaj tiv thaiv: protein carbonyl thawj antibody (Anti-DNP; # STA-308, Cell Biolabs) thiab SOD2 (#Ab13533, Abcam). Tom qab peb 10 feeb ntxuav hauv TBST, daim nyias nyias tau soj ntsuam nrog cov tshuaj tiv thaiv thib ob rau cov protein carbonyl (HRP-conjugate; # STA-308, Cell Biolabs) thiab SOD2 (tiv thaiv luav IgG-HRP txuas; # 7074, Cell Signalling) hauv kev thaiv cov tshuaj rau 2 h ntawm chav tsev kub thiab thaum kawg tau ntxuav nrog TBST. Lub dilution ntawm thawj thiab theem nrab cov tshuaj tiv thaiv tau ua kom zoo rau txhua qhov tshuaj tiv thaiv. Kev tshawb pom Chemiluminescence tau ua tiav siv ECL Clarity (Biorad), thiab cov duab ntes tau ua tiav nrog ChemiDocMP. Tag nrho cov duab tau txheeb xyuas siv Image Lab 4.0.1 software. Normalization ntawm cov teeb liab cov teeb liab siv tau ua raws li qhov muaj pes tsawg leeg ntawm tag nrho cov protein ntau ntawm SF cov duab, qauv tswj, thiab cov qauv sab hauv.

2.10. NAD Measurement

Tag nrho NAD thiab NADH qib tau ntsuas hauv homogenized dawb gastrocnemius siv cov khoom siv coj mus muag (#K337-100, Biovision, USA) thiab ua raws li cov chaw tsim khoom cov lus qhia. Luv luv, tag nrho cov pas dej ntawm NAD (NADt= NADt thiab NADH) raug rho tawm. Rau txhua tus qauv muab rho tawm, ib nrab ntawm cov qauv tau muab rhaub rau 60 degree rau 30min kom decompose NADt thaum khaws NADH kom zoo. Ob qho NADt thiab NADH cov qauv tau sib xyaw nrog NAD cycling enzyme, thiab nqus tau ntsuas ntawm 450nm. NADt thiab NADH raug ntsuas los ntawm kev sib piv nrog NADH tus qauv nkhaus thiab normalizing rau mg ntawm cov protein. Thaum kawg, qhov piv ntawm NAD ntxiv (NADt-NADH) rau NADH raug xam. Tag nrho cov kev ntsuam xyuas tau ua nyob rau hauv triplicate.

2.11. Kev txheeb cais

Cov ntaub ntawv raug tshaj tawm raws li txhais tau tias ± tus qauv sib txawv (SD). Cov ntaub ntawv normality raug soj ntsuam siv D'Agostino-Pearson normality test. Kev tshuaj xyuas cov ntaub ntawv tau ua los ntawm Cov Tub Ntxhais Kawm T-test thiab ob-txoj kev ANOVA (pab pawg thiab sijhawm) rau kev ntsuas rov qab los ntawm Bonferroni qhov kev xeem hoc siv Prism8.4.1(GraphPad Software, San Diego, CA, USA). Ib theem ob sab ntawm 5 feem pua ​​​​rau hom I yuam kev raug siv.

3. Cov txiaj ntsig

3.1. Lub cev muaj pes tsawg leeg thiab tag nrho lub cev Metabolism

Kaum tsib hnub ntawm CF supplementation, nyob rau hauv qus-hom (WT) nas, tsis hloov lub cev hnyav (p=0.091), lean mass(p=0.{{24} }73) thiab rog rog ntsuas los ntawm EchoMRI (p=0.89; Daim duab 1A). O2 noj cov nqi qhia ib ntshiv loj tau ntau dua nyob rau hauv lub voj voog tsaus nti (8 ± 8 feem pua ​​​​, p=0 042), hos nws tseem zoo ib yam nyob rau hauv lub teeb voj voog (p =0 .392; Daim duab 1B.). Tsis muaj kev hloov pauv hauv kev ua haujlwm hauv locomotor (cov ntaub ntawv tsis qhia). CF nas pom qhov nce ntxiv ntawm tag nrho lub cev siv zog los ntawm 15 ± 15 feem pua ​​(p =0.042; Daim duab 1C) thiab hauv noj zaub mov (0.08±0.01 vs.0.11±0.01 g ib lub cev hnyav, raws li CF pawg thiab WT, p=0.013, Daim duab 1D).

CF supplementation tau hloov pauv cov metabolism hauv lub cev tag nrho: cov txiaj ntsig tseem ceeb ntawm RER siab dua hauv CF pawg tau pom dua 24 h (p=0.023, Daim duab 2A) thiab txhais tau tias RER ntau dua 12 teev siab dua nyob rau hauv CF pawg nyob rau hauv lub teeb thiab tsaus voj voog (ntxiv rau 5 ± 3 feem pua, p=0. Ntxiv mus, CF supplementation txhawb nqa carbohydrate (CHO) siv (51 ± 13 feem pua ​​​​ntawm kev siv hluav taws xob nyob ntawm CHO oxidation hauv CF pawg vs.37 ± 12 feem pua ​​​​hauv pab pawg tswj thaum lub teeb ci, p=0.108 thiab 71 ± 21 feem pua ​​​​hauv CF pawg vs.47 ± 24 feem pua ​​​​hauv pawg tswj hwm thaum lub voj voog tsaus, p=0.004; Daim duab 2B.). Txawm li cas los xij, thaum qhia hauv kJ, tsis muaj qhov sib txawv hauv CHO oxidation lossis fatty acid oxidation (CHOoxidation: 3.7 ± 1.4 kcal·d-1 hauv kev tswj hwm vs.4.4 ± 1.7 kcal.lean lub cev hnyav -1. d-1 hauv CF, p=0.33 thiab fatty acid oxidation: 2.3 ± 1.0 kcal·d-Iin tswj vs.2.6±0.87 kcal.lean lub cev hnyav-1.d{{ 50}}hauv CF,p=0.608).Kev soj ntsuam ntawm PRCF ntawm RER qhia qhov nce ntawm EC50 qhov tseem ceeb hauv pawg CF (p=0,009; Daim duab 2D) txhais tau tias CF ua rau muaj kev hloov pauv mus rau kev siv ntau dua ntawm carbohydrates. Qhov txo qis ntawm txoj kab nqes hav tau pom hauv pawg CF (H qhov tseem ceeb: 25.16 ± 4.44 hauv kev tswj hwm vs.12.79 ± 3.96 hauv pawg CF, p<0.001). moreover,="" the="" rer="" amplitude="" over="" 24h="" was="" increased="" by="" 31±="" 31%="" in="" the="" cf="" group="" (p="0.035)." cf="" supplementation="" improved="" glucose="" tolerance="" following="" oral="" glucose="" administration="" from="" unchanged="" baseline="" blood="" glucose="" levels="" after="" an="" overnight="" fast="" (figure="" 2e,p="0.003)." the="" area="" under="" the="" capillary="" blood="" glucose="" curve="" was="" lower="" in="" the="" cf="" group(1018±135="" vs.="" 1181±="" 170="" mm·120="" min,="" p="0.005;" figure="">

3.2. Mitochondrial Bioenergetics

Peb tau soj ntsuam cov nyhuv ntawm CF supplementation ntawm lub zog ntawm mitochondria nyob rau hauv lub dawb gastrocnemius thiab soleus nqaij nyob rau hauv WT nas. Kev ua pa siab dua mitochondrial siv cov complex IV substrates hauv cov nqaij dawb gastrocnemius thiab soleus cov leeg tau pom (p=0.004 thiab p=0.028, ntsig txog; Daim duab 3A, B). Tsis muaj kev hloov pauv ntawm CII / CI thiab CIV / CI ua pa piv tau raug soj ntsuam (p =0.899 thiab p =0.701 rau cov kab mob dawb gastrocnemius thiab p=0.693 thiab p{{ 11}}.912 rau cov leeg nqaij soleus, ntsig txog, cov ntaub ntawv tsis qhia). Ntxiv mus, complex, I, IV, thiab citrate synthase kev ua ub no tau nce ntau hauv cov nqaij dawb gastrocnemius (feem ntau: ntxiv rau 31 ± 27 feem pua, p=0.004; ntxiv rau 28 ± 38 feem pua, p=0 .027; ntxiv rau 35 ± 38 feem pua, p= 0.009; ntxiv rau 14 ± 13 feem pua, p=0.040; Daim duab 3C).Lub peev xwm ntawm mitochondria rau oxidize palmitoyl-carnitine nyhav nce nyob rau hauv cov dawb gastrocnemius (ntxiv rau 24 ± 12 feem pua ​​​​, p =0.096; Daim duab 3D), whereas tsis muaj qhov sib txawv ntawm cov soleus (p =0.363). Cov txheeb ze tus nqi ntawm palmitoyl-carnitine-stimulated ua pa tsis hloov pauv thaum qhia hauv kev ua haujlwm ntawm kev ua pa nyuaj (62 ± 11 hauv pawg tswj hwm vs.64 ± 20 hauv pawg CF hauv cov kab mob dawb gastrocnemius thiab 70 ± 18 vs.72 ± 16 nyob rau hauv lub soleus). Tom ntej no, peb tshawb nrhiav qhov rhiab heev rau Ca ntxiv rau -induced PTP qhib nyob rau hauv cov dawb gastrocnemius. Raws li pom hauv daim duab 3E, tsis yog lub sijhawm rau PTP qhib lossis lub peev xwm Ca²retention tau hloov kho tom qab CF supplementation (p=0.608 thiab p=0.943, feem).

mRNA kev ntsuam xyuas ntawm cov noob koom nrog hauv mitochondrial biogenesis pom 70 feem pua ​​​​upregulation ntawm NRF1 mRNA, qhov tsis sib txawv tau tshaj tawm rau PGCl, Tfam, CS, ND1, ND2, SDHa thiab Cox2 (Table 3). Peb tshawb nrhiav, ntawm ob peb cov qauv, cov txiaj ntsig ntawm CF supplementation ntawm mitochondrial supercomplexes (Daim duab S1, Cov Khoom Siv Ntxiv). Kev tsom xam densitometric qhia tias CF supplementation tau nce tag nrho cov ntsiab lus supercomplexes yam tsis muaj kev hloov pauv zoo lossis hloov kho. Qhov tseeb, kev ua pa ceev tau txhim kho los ntawm 58 feem pua ​​​​ntawm kev siv cov complex I thiab complexⅡ sojntsuam thiab los ntawm 93 feem pua ​​​​ntawm kev siv complex I sojntsuam. Ib yam li ntawd, complex I thiab complex III cov ntsiab lus embedded hauv supercomplexes tau siab dua hauv CF pawg.

CF supplementation txo qis mitochondrial ROS emission. Thaum H2O2 emission tseem tsis hloov pauv ob qho tib si nyob rau hauv cov dawb gastrocnemius thiab soleus thaum qhia ib qhov hnyav qhuav (Daim duab 4A, B, raws li), cov nqi tau txo qis thaum qhia ib cov ntsiab lus mitochondrial (Daim duab 4C). Lub ntsiab zoo ntawm CF supplementation tau pom nyob rau hauv ntau yam mob nyob rau hauv cov dawb gastrocnemius (p<0.048; figure="" 4c).="" the="" mitochondrial="" h2o2="" emission="" was="" also="" significantly="" reduced="" when="" expressed="" peroxidative="" phosphorylation="" capacity="" assessed="" by="" state="" 4="" respiration="" using="" complex="" i+ii="" substrates="" by="" 26±13%="" in="" the="" white="" gastrocnemius="" (p="0.044)and" by="" 29±12%="" in="" the="" soleus(p="0.024;" figure="">

3.3. Oxidative Stress Markers nyob rau hauv Skeletal Muscle

Cov cellular protein oxidative kev ntxhov siab nyob rau hauv WT nas tsis cuam tshuam los ntawm CF supple-mentation raws li cov cellular cov ntsiab lus ntawm SOD2 zoo ib yam nyob rau hauv cov kev sim thiab tswj pab pawg nyob rau hauv lub dawb gastrocnemius thiab soleus (Daim duab 4E). Ntxiv mus, tsis muaj qhov sib txawv tau pom nyob rau hauv cov protein carbonylation nyob rau hauv cov dawb gastrocnemius thiab soleus (Daim duab 4F). Tsis tas li ntawd, tsis muaj qhov sib txawv ntawm catalase thiab MnSod mRNA qhia tau pom tom qab CF supplementation (Table 3).

3.4.NAD Metabolism

Tag nrho cov pas dej ua ke ntawm pyridine nucleotides nce tom qab CF supplementation los ntawm 36 ± 33 feem pua ​​(p=0.012).Tshwj xeeb, NAD thiab NADH cov ntsiab lus tau nce hauv cov kab mob plab dawb (feem ntau los ntawm 69 ± 60 feem pua, p{{6 }}.026 thiab 29 ± 42 feem pua, p=0.017; Daim duab 5A,B) thiab NAD ntxiv / NADH piv tau nce ntxiv hauv pawg CF (p =0.084).No Cov txiaj ntsig tseem ceeb tau pom rau Sirt3 mRNA (p=0.096), NMNAT mRNA (p=0.094), thiab Sirt1 mRNA(p =0.869; Table 3 ).

3.5.Txhua lub cev thiab Cellular Metabolism teb Tom qab CF Supplementation hauv Sirt3-/ Cov nas

Peb tau tshuaj xyuas cov txiaj ntsig ntawm CF supplementation hauv Sirt{{0}} nas. Lub cev muaj pes tsawg leeg raws li qhov hnyav, hnyav hnyav, thiab rog rog tsis txawv ntawm CF thiab pawg tswj hwm (p= 0.52,p=0.66,p=0.45, feem; Daim duab 6A). Ib yam li ntawd, kev siv hluav taws xob niaj hnub tsis cuam tshuam los ntawm CF supplementation ({{10}}.38±{16}}.{{40}}1 Kcal.lean lub cev huab hwm coj-1.hnub-1 hauv kev tswj hwm vs. 0.39 ± 0.01 Kcal·lean lub cev hnyav-1.hnub-1 hauv CF, p=0.502; Daim duab 6B). Lub RER zoo ib yam ntawm ob pawg hauv lub teeb thiab tsaus voj voog (p=0.301; Daim duab 6C), thiab substrate oxidation tsis cuam tshuam los ntawm CF supplementation (44 ± 17 feem pua ​​​​ntawm lub zog noj nyob ntawm CHO oxidation hauv pawg tswj vs. 50 ± 11 feem pua ​​​​hauv pawg CF thaum lub sij hawm lub teeb theem, p=0.209 thiab 65 ± 19 feem pua ​​​​hauv pawg tswj vs.77 ± 21 feem pua ​​​​hauv CF pawg thaum lub sijhawm tsaus ntuj, p=0.395; Daim duab 6D). Cov metabolic yooj tau soj ntsuam los ntawm RER amplitude tshaj 24 h tseem zoo sib xws ntawm ob pawg (0.19 ± 0.05 hauv kev tswj hwm vs.0.20 ± 0.03 hauv CF, p=0.604), whereas PRCF txoj kab nqes tau txo qis hauv pawg CF. (H qhov tseem ceeb: 17.77 ± 4.38 hauv kev tswj hwm vs.12.79 ± 3.93 hauv CF, p=0.039).

Tom ntej no, peb piv cov nyhuv ntawm CF supplementation ntawm mitochondrial enzyme kev ua ub no. CF supplementation ua tsis tau tejyam ua kom cov enzyme kev ua ntawm complex I, I, IV, thiab CS nyob rau hauv Sirt3- / nas raws li tau pom yav dhau los hauv WT (raws li: p=0.623, p=0 .629,p=0.283, thiab p=0.791, Daim duab 6E).Great enzyme kev ua ntawm complex thiab IV tau pom nyob rau hauv WT piv rau Sirt3-7 nas tom qab CF supplementation ( raws li: p=0.027 thiab P=0.045)

4. Kev sib tham

Hauv qhov kev tshawb fawb no, peb muab kev txheeb xyuas kev sib koom ua ke ntawm cov txiaj ntsig ntawm CF ingestion. Peb tau pom tias kev noj CF mob ntev tau txhim kho mitochondrial ua pa thiab txo qis H2O2 ntau lawm, soj ntsuam hauv lawv qhov chaw myofibre, qhov kev cuam tshuam ntawm PTP qhib tsis hloov pauv. CF ingestion txhim khu NADmetabolism, txhawb kev koom tes ntawm sirtuin txoj hauv kev pom mitochondrial adaptations. Peb kuj tau soj ntsuam tag nrho lub cev metabolism hloov pauv mus rau lub peev xwm ntau dua los siv cov carbohydrates ua lub ntsiab substrate qhia tias mitochondrial adaptations yuav ua rau pom tag nrho lub cev metabolism hloov.

4.1.Metabolism

Kev tswj hwm kev noj zaub mov ntawm cocoa flavanols tau raug txheeb xyuas tias yog lub tswv yim zoo rau kev txo cov piam thaj tsis haum, tso cai rau kev kho cov kab mob metabolic [34]. Peb cov txiaj ntsig tau ua raws li cov kev tshawb fawb yav dhau los uas tau tshaj tawm tias muaj cov piam thaj ntau ntxiv tom qab noj flavanols [35,36]. Cov txheej txheem npaj rau qhov kev tshwm sim no suav nrog kev nce hauv kev hloov pauv ntawm GLUT4 rau ntawm cov cell membrane, nce hauv phosphorylation ntawm AMPK, thiab kev tswj hwm ntawm UCP-2 gene qhia hauv cov leeg pob txha [8,37] . Tsis tas li ntawd, kev noj CF yog txuam nrog kev txhim kho cov tshuaj insulin hauv tib neeg [8]. Tom qab 15 hnub ntawm kev noj cov qhob noom xim kasfes tsaus, qhov txo qis ntawm homeostasis qauv kev ntsuam xyuas ntawm cov tshuaj insulin kuj tau pom nyob rau hauv cov neeg noj qab haus huv nrog rau qhov nce ntawm qhov ntau ntawm cov tshuaj insulin rhiab heev thiab cov tshuaj insulin rhiab heev [8]. Peb kuj tau pom ib qho kev nce ntxiv hauv cov piam thaj hauv cov ntshav thiab cov metabolic yooj yooj yim hauv WT nas, uas yog hais txog lub cev muaj peev xwm los hloov cov roj oxidation rau roj muaj [38]. Interestingly, thaum lub sij hawm impaired mitochondrial oxidative muaj peev xwm thiab lwm yam substrate oxidation yog txuam nrog insulin tsis kam 39, endurance ce kev cob qhia nce mitochondrial cov ntsiab lus, insulin rhiab heev, thiab metabolic yooj [38]. Yog li, cov txiaj ntsig tam sim no yog tshwj xeeb rau CF supplementation thiab tsis cuam tshuam los ntawm kev ua haujlwm.

Cov txiaj ntsig zoo ntawm CF ntawm kev hloov pauv hauv metabolic, nrog rau qhov nce ntawm mitochondrial complexes kev ua haujlwm, tau blunted hauv Sirt3-7 nas, qhia tias qhov zoo ntawm CF tshwm sim tsawg kawg ntawm Sirt3. Cov txiaj ntsig no txhawb nqa kev tshawb pom yav dhau los thiab lub luag haujlwm tseem ceeb ntawm Sirt3 ntawm kev tswj hwm kev hloov pauv hauv metabolic [24]. Peb cov txiaj ntsig tau qhia tias qhov nce hauv mitochondrial muaj nuj nqi tuaj yeem txhim kho metabolic yooj. Txawm li cas los xij, nrog rau kev ua haujlwm ntawm mitochondrial, lub basal respiratory quotient, qabzib pov tseg tus nqi, adipose ntaub so ntswg lipid cia muaj peev xwm, thiab plasma dawb fatty acid concentration kuj tau txheeb xyuas raws li qhov tseem ceeb ntawm metabolic yooj thiab yuav tsum tau txiav txim siab [40]. Cov kev tshawb fawb yav tom ntej yuav tsum tau soj ntsuam cov cellular determinants ntawm metabolic yooj yooj yim dua kom tob rau peb nkag siab txog cov hauv paus ntsiab lus ntawm kev txhim kho ntawm metabolic yooj tom qab CF supplementation.

Kev ntsuam xyuas tag nrho lub cev metabolism tau nthuav tawm qhov nce ntawm tus txheeb ze CHO oxidation tom qab CF supplementation. Cov txiaj ntsig no sib piv nrog cov kev tshawb fawb yav dhau los uas tau tshaj tawm txog kev nce lipolysis tom qab noj flavanols mob hnyav lossis ntxiv ob lub lis piam ntxiv nrog flavan-3-ols hauv nas [36]. Dhau li ntawm qhov sib txawv ntawm qhov sib txawv ntawm qhov sib txawv, cov kev pom tsis sib haum xeeb no yuav tshwm sim los ntawm cov teebmeem pleiotropic ntawm NO. Nws tau txais txiaj ntsig zoo tias kev noj CF txhawb kev tsim khoom lossis bioavailability ntawm NO metabolism [7]. Ntawm ib sab, TSIS txhawb nqa cov piam thaj thauj thiab nce hauv cov leeg pob txha. Kev ua kom tsis muaj kev tsim tawm hauv cov xov tooj ntawm tes-los ntawm myotubes los ntawm insulin lossis hydrogen peroxide ua rau muaj qhov nce ntawm GLUT4 translocation, uas tau txo nrog nNOS inhibition [41]. Ntxiv mus, TSIS TAU tuaj yeem nce qabzib thauj mus rau hauv txoj hauv kev ywj pheej ntawm insulin uas ua rau nce qib ntawm cyclic GMP thiab AMPK [42], uas txhawb nqa cov piam thaj oxidation hauv cov leeg pob txha ntawm cov leeg nqaij tsis muaj fatty acid thiab oxidation [43]. Ntawm qhov tod tes, TSIS txhawb cov fatty acid oxidation los ntawm kev txo qis ntawm malonyl-CoA ntawm inhibition ntawm acetyl-CoA carboxylase thiab ua kom cov malonyl-CoA decarboxylase hauv cov leeg pob txha [44]. Thaum peb tau pom qhov hloov pauv mus rau qhov sib piv qis dua los siv fatty acid li substrate nrog CF, tsis muaj qhov sib txawv ntawm qhov tseem ceeb hauv kJ. Qhov no qhia tau hais tias qhov nce hauv kev siv hluav taws xob tau pom nrog CF supplementation nyob ntawm kev nce hauv cov piam thaj oxidation. Yog li, cov txiaj ntsig no txhawb lub tswv yim tias kev noj CF nce insulin rhiab heev thiab txhawb nqa cov piam thaj oxidation yam tsis muaj kev cuam tshuam ntawm fatty acid oxidation muaj peev xwm. Cov txiaj ntsig no tsa cov lus nug ntawm qhov cuam tshuam rau kev siv cocoa flavanols hauv cov ntsiab lus ntawm kev noj zaub mov muaj roj. Cov kev tshawb fawb yav tom ntej yuav tsum ntsuas qhov ua rau muaj kev phom sij ntawm kev koom tes ntawm CF supplementation thiab kev noj zaub mov muaj roj.

4.2.Mitochondrial Function

Kev noj haus ntawm cocoa flavanols tau pom tias txhim kho mitochondrial muaj nuj nqi, suav nrog (i) mitochondrial respiration thiab electron thauj saw enzyme kev ua ub no; (i) mitochondrial H2O2 tso tawm, uas qhia txog mitochondrial reactive oxygen hom (ROS) ntau lawm; thiab (i) mitochondrial calcium retention muaj peev xwm uas qhia txog qhov cuam tshuam rau mitochondrial permeability transition pore (mPTP) qhib (saib xyuas saib [11]. CF supplementation ntawm mitochondrial muaj nuj nqi soj ntsuam nyob rau hauv situ nyob rau hauv permeabilized fiber ntau thiab nyob rau hauv sib txawv cov leeg nqaij. kev cuam tshuam thaum lub sij hawm mitochondrial kev cais tawm [32]. Yog li ntawd, nws yog ib qho tseem ceeb uas yuav tau kawm txog mitochondrial muaj nuj nqi hauv cov ntaub so ntswg npaj qhov twg cov qauv mitochondrial tau khaws cia, thiab tag nrho cov pas dej mitochondrial yog sawv cev los soj ntsuam cov lus teb rau kev kho mob zoo dua.

Cov kev tshawb fawb yav dhau los tau ua hauv kab lis kev cai ntawm tes lossis hauv vivo hauv cov nas tau pom tias kev ntxiv nrog EPI, uas yog suav tias yog lub ntsiab bioactive molecule hauv CF, nce siab tshaj plaws ADP-stimulated ua pa (xws li, xeev 3 ua pa) thaum mitochondria muaj zog nrog kev sib xyaw ntawm lub zog. substrates pub ntau qhov chaw raws cov kab ua pa [45-49]. Raws li cov txiaj ntsig no, peb pom cov txiaj ntsig tseem ceeb ntawm CF supplementation ntawm mitochondrial ua pa hauv cov leeg oxidative thiab glycolytic. Txhawm rau txiav txim siab seb qhov kev nce ntxiv no cuam tshuam li cas rau qhov nce ntawm mitochondrial lossis kev hloov kho ntawm mitochondrial respiratory complexes, peb tau soj ntsuam mitochondrial supercomplexes los ntawm cov leeg quadriceps. Cov qauv supramolecular no tau xav tias yuav txo qis ROS ntau lawm, ruaj khov lossis pab hauv kev sib dhos ntawm cov neeg ua haujlwm, tswj cov kab mob ua pa, thiab tiv thaiv kev sib sau ua ke ntawm cov protein ntau hauv cov protein ntau hauv mitochondrial membrane [50-52]. Txawm hais tias muaj qee qhov kev ntsuas, qhov kev tsom xam pom ntawm cov txiaj ntsig (Cov Khoom Siv Ntxiv) qhia tias CF supplementation induces ib qho kev nce ntawm mitochondrial loj, raws li qhov tshwm sim los ntawm kev nce ntawm txhua hom supercomplex, es tsis yog cov kab mob ua pa tshwj xeeb. Sib sau ua ke, cov txiaj ntsig no qhia tias CF txhawb nqa lub mitochondrial loj zuj zus yam tsis muaj kev hloov pauv ntawm cov kab mob ua pa nyuaj.

Thaum lub sij hawm mitochondrial ua pa, qhov sib txawv ntawm cov superoxide yog tsim thiab tuaj yeem metabolized los ua lwm hom ROS [53]. Txawm hais tias ntau dhau ROS ntau lawm los ntawm mitochondria tau koom nrog ntau yam ntawm pathologies, tsis tshua muaj lub cev muaj zog ntawm ROS tuaj yeem muaj txiaj ntsig zoo, xws li tiv thaiv cov kab mob sib kis thiab koom nrog hauv xov tooj ntawm tes [2,54]. Cov kev tshawb fawb yav dhau los tau ua nyob rau hauv vivo hauv cov nas thiab tib neeg thiab cov qauv kab lis kev cai ntawm tes tau tshaj tawm cov txiaj ntsig CF supplementation los ntawm ROS ntau lawm thiab ntau lub tshuab antioxidant [11]. Piv txwv li, qhov ncauj gavage nrog EPIduring 15 hnub nce SOD2 thiab catalase kev ua hauv nas quadriceps cov leeg [17]. Thaum mitochondrial ROS ntau lawm tsis tau txais kev soj ntsuam dav dav, peb pom tias ROS cov qib ntau lawm zoo ib yam thaum cov txiaj ntsig tau nthuav tawm ntawm cov leeg nqaij, qhov kev tsim tawm tau qis dua thaum hais txog ib qho mitochondrial loj, qhia tias kev hloov pauv zoo tshwm sim los tswj lub cev muaj zog ntawm ROS. Ntxiv mus, peb tsis tau soj ntsuam ib qho kev cuam tshuam ntawm CF ntawm SOD2 lossis ROS-vim kev puas tsuaj los ntawm cov protein carbonylation. Thaum CF txhim kho cov pas dej antioxidant antioxidant, suav nrog kev tshaj tawm SOD2 thiab catalase qib [16,17,55], qhov kev hloov pauv no tsis yog ib txwm cuam tshuam nrog kev txo qis hauv ROS vim kev puas tsuaj [56-59]. Cov ntaub ntawv no txhawb txoj kev xav tias qib qis ntawm ROS ntau lawm yog qhov yuav tsum tau muaj rau cov cell homeostasis [2]. Sib sau ua ke, cov txiaj ntsig no qhia tias, ua ke kom nce ntawm mitochondrial loj, CF txhawb nqa cov kev hloov pauv ntawm mitochondrial los txhim kho nws cov peev xwm antioxidant thiab tswj cov qib qis ntawm ROS ntau lawm.

4.3.Mitochondrial Biogenesis

Mitochondrial loj yog qhov tshwm sim ntawm kev sib cuam tshuam ntawm mitochondrial biogenesis thiab mitophagy. Peb txoj kev tshawb fawb tsom mus rau mitochondrial biogenesis qhib los ntawm polyphenols noj [60]. Mitochondrial biogenesis yog cov txheej txheem cellular uas txhim kho mitochondrial loj. Cov txheej txheem nyuaj thiab tswj tau zoo no suav nrog ntau yam kev hloov pauv, cov tshuaj hormones nuclear receptors, thiab cov coactivators transcription uas ua ke los tswj cov kev hloov pauv hauv kev qhia ntawm nuclear thiab mitochondrial DNA encoded noob [61]. Cov kev tshawb fawb yav dhau los tau sim txheeb xyuas txoj hauv kev twg tuaj yeem ua rau CF supplementation-induced mitochondrial biogenesis. Kev txhawb nqa ntawm NO-dependent signaling tau tshwm sim raws li txoj hauv kev hauv nruab nrab koom nrog hauv mitochondrial biogenesis. CF supplementation txhim kho cov tiam ntawm NO los ntawm inhibiting arginine degrading enzyme arginase, uas ua rau kom muaj L-arginine rau TSIS biosynthesis [62,63]. Ntxiv mus, siv eNOS inhibitor, cov nyhuv stimulatory ntawm epicatechin ntawm mitochondrial biogenesis yog ib feem blunted [17]. Cov txiaj ntsig no qhia txog kev koom tes ntawm lwm txoj hauv kev taw qhia los txhawb mitochondrial biogenesis.

Tsis ntev los no, Aragones et al. (2016) tau tshaj tawm tias kev noj zaub mov proanthocyanidins, cov tswv cuab ntawm tsev neeg polyphenols, txhawb cov kab mob siab NAD ntxiv rau cov metabolism los ntawm kev txhim kho cov de tam sim no o NAD ntxiv rau txoj kev biosynthesis thiab Sirte kev ua si nyob rau hauv ib koob tshuaj nyob rau hauv nas[12]. Tsis tas li ntawd, lub siab xa NADt ua ntej mus rau lwm lub cev, xws li cov leeg pob txha, uas muaj peev xwm qis dua los ua ke NAD, ua rau muaj peev xwm nthuav tawm cov teebmeem tshwm sim hauv daim siab rau tag nrho cov kabmob [64]. Thaum qee qhov kev tshawb fawb tau tshaj tawm txog kev cuam tshuam ntawm CF supplementation ntawm Sirt1 protein cov ntsiab lus, kev ua haujlwm, lossis mRNA [12,15-17,65,66], kev koom tes ntawm NAD metabolism hauv mitochondrial biogenesis tom qab CF supple-mentation siv poob lossis nce ntawm function yeej tsis tau sim. Thaum lub cev nqaij daim tawv tshwj xeeb khob tawm yuav zoo dua rau tag nrho lub cev Sirt3 KO nas siv hauv peb txoj kev tshawb fawb, peb cov txiaj ntsig tau qhia tias CF txhim kho NAD metabolism, thiab Sirt3 tau koom nrog hauv qhov kev txhawb nqa no-induced mitochondrial electron thauj saw kev txhim kho. Cov txiaj ntsig zoo tau pom ntawm kev ua haujlwm ntawm mitochondrial complexes tom qab CF supple-mentation tsis tau pom siv Sirt3-7 nas thiab ntau dua ntawm cov haujlwm I thiab IV kev ua haujlwm tau pom tom qab CF supplementation hauv WT nkaus xwb. Cov kev soj ntsuam no txuas ntxiv ua haujlwm dhau los qhia tias Sirt3 ua lub luag haujlwm tseem ceeb hauv kev ua haujlwm mitochondrial]67. Sib sau ua ke, cov kev tshawb pom no qhia tias sirtuins, los ntawm kev hloov kho ntawm NAD metabolism, koom nrog hauv mitochondrial loj txhim kho pom tom qab CF supplementation. 4.4.Limits

Cocoa taum yog tsim los ntawm kev sib xyaw ntawm monomeric, oligomeric, thiab polymeric flavanols. Ntawm cov flavanols, EPI zoo li yog cov polyphenolic monomer tshaj plaws nyob rau hauv cov khoom cacao, sawv cev txog li 35 feem pua ​​​​ntawm cov ntsiab lus polyphenol [68]. EPI tau tsim los ua lub ntsiab bioactive molecule hauv qab cov txiaj ntsig cuam tshuam nrog cocoa thiab chocolate supplementation [47,69]. Txawm li cas los xij, EPI metabolites (EPIm) tuaj yeem koom nrog EPI-induced biological teebmeem pom hauv vivo kev tshawb fawb. EPIm tuaj yeem ncav cuag plasma ntau dua li EPI [70]. Ntxiv mus, qhov sib txawv loj hauv EPIm tau pom nyob thoob plaws hom. Tseeb tiag, 80 feem pua ​​​​ntawm EPIm tam sim no hauv tib neeg tsis tau kuaj pom hauv nas, thaum qhov zoo sib xws ntawm nas thiab tib neeg tau pom tias muaj qhov zoo dua me ntsis vim tias ob qho tib neeg metabolites loj tau kuaj pom [70]. Peb siv enriched CF extract uas muaj txog li tsib zaug EPI ntau dua unfermented cocoa taum[71]. Ntxiv mus, EPI cov ntsiab lus raug txo los ntawm 10-20 feem pua ​​​​los ntawm fermentation thiab txo qis hauv qhov kub thiab txias thaum lub sij hawm ci [71,72]. Ua ke, cov ntaub ntawv no qhia tias tsuas yog yuav tsum tau them nyiaj rau EPI cov ntsiab lus thaum soj ntsuam cov txiaj ntsig ntawm EPI ntxiv rau tib neeg kev noj qab haus huv, thiab cov txiaj ntsig tam sim no yuav tsum tau txiav txim siab ceev faj ua ntej ntxiv cov txheej txheem pom hauv tib neeg.

5. Cov lus xaus

Txhua hnub CF supplementation rau 15 hnub ua rau mitochondrial muaj nuj nqi txhim kho hauv cov leeg oxidative thiab glycolytic. Cov txiaj ntsig tam sim no qhia tau hais tias qhov nce mitochondrial ua pa ua rau muaj kev nce hauv mitochondrial huab hwm coj thaum lub cellular theem ntawm ROS ntau lawm tau khaws cia, qhia txog kev hloov pauv zoo hauv mitochondria. Concomitant rau cov nyhuv cellular ntawm NO metabolism yav dhau los tau piav qhia [73], CF modulates NAD metabolism, uas txhawb kev ua sirtuins. Sirt3 plays lub luag haujlwm tseem ceeb hauv mitochondrial adaptations thiab metabolic yooj tom qab CF supplementation. Raws li sirtuins yog lub hom phiaj tsis ntev los no nyob rau hauv ntau yam kab mob pathogenesis, xws li ntshav qab zib lossis mob hawb pob [74,75l, cov kev tshawb fawb ntxiv yuav tshawb txog qhov kev txaus siab ntawm kev siv CF raws li lub ntuj activator ntawm sirtuins.

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