Randomized Trial : D-Glyceric Acid Activates Mitochondrial Metabolism in 50–60-Year-Old Healthy Humans

This is a sel -a chi ed e sion o an o iginal a icle. This e sion may di e om he o iginal in pagina ion and ypog aphic de ails. Au ho (s): Ti le: Yea : Ve sion: Copy igh : Righ s: Righ s u l: Please ci e he o iginal e sion: CC BY 4.0 h ps://c ea i ecommons.o g/licenses/by/4.0/ Randomized T ial : D-Glyce ic Acid Ac i a es Mi ochond ial Me abolism in 50–60-Yea - Old Heal hy Humans © 2021 Hi onen, Ky öläinen, Leh i and Kainulainen. Published e sion Hi onen, O. Pe e i; Ky öläinen, Heikki; Leh i, Maa i ; Kainulainen, Heikki Hi onen, O. P., Ky öläinen, H., Leh i, M., & Kainulainen, H. (2021). Randomized T ial : D-Glyce ic Acid Ac i a es Mi ochond ial Me abolism in 50–60-Yea -Old Heal hy Humans. F on ie s in Aging, 2, A icle 752636. h ps://doi.o g/10.3389/ agi.2021.752636 2021 Randomized T ial: D-Glyce ic Acid Ac i a es Mi ochond ial Me abolism in 50–60-Yea -Old Heal hy Humans O. Pe e i Hi onen*, Heikki Ky öläinen, Maa i Leh i and Heikki Kainulainen Facul y o Spo and Heal h Sciences, Neu omuscula Resea ch Cen e , Uni e si y o Jy äskylä, Jy äskylä, Finland Backg ound: Based on ea lie s udies, na u al me aboli e D-glyce ic acid (DGA) does no seem o play any ole in whole-body me abolism. Ne e heless, one e hanol oxida ion- ela ed a s udy wi h con o e sial esul s aised ou in e es . Acco ding o p epa a o y s udies o he egula o y app o al o DGA, some highly conse ed mechanism seems o sub ly ac i a e he cellula ene gy me abolism. The e o e, he p esen 25-days double- blind human s udy wi h placebo con ol was ini ia ed. Pu pose: The main a ge in he p esen s udy wi h 27 heal hy 50–60-yea -old human olun ee s was o find ou whe he an “acu e”4-days and a longe 21-days exogenous DGA egimen caused mode a e ac i a ion o he mi ochond ial ene gy me abolism. The simul aneous a ge was o find ou whe he a hal ed dose o DGA con inued o be an e ec i e egimen. Main Findings: The esul s e ealed he ollowing s a is ically significan findings: 1) plasma concen a ions o me aboli es ela ed o ae obic ene gy p oduc ion, especially lac a e, we e s ongly educed, 2) sys emic inflamma ion was lowe ed bo h in 4- and 21- days, 3) mi ochond ia- ela ed mRNA exp essions in ci cula ing immune cells we e no iceably modula ed a Day4, 4) cellula memb ane in eg i y seemed o be sha ply enhanced, and 5) cellula NADH/NAD + - a io was up egula ed. Conclusion: Mi ochond ial me abolism was clea ly up egula ed a he whole-body le el in bo h 4- and 21 days. A he same ime, he e ec o DGA was e y well ole a ed. Based on ecei ed solid esul s, he DGA egimen may alle ia e acu e and ch onic ene gy me abolic challenges in main o gans like he li e , CNS, and skele al muscles. Enhanced memb ane in eg i y combined wi h lowe sys emic inflamma ion and ac i a ed me abolic flows by he DGA egimen may be beneficial especially o he aging popula ion. Keywo ds: mi ochond ial ac i a ion, e-oxida ion, memb ane in eg i y, subclinical inflamma ion, DGA ac i a ion Edi ed by: Smijin K Soman, Uni e si y o Ne ada, Uni ed S a es Re iewed by: Ting Liu, Johns Hopkins Uni e si y, Uni ed S a es Julie Reisz Haines, Uni e si y o Colo ado Den e , Uni ed S a es *Co espondence: O. Pe e i Hi onen [email p o ec ed].fi Special y sec ion: This a icle was submi ed o Aging, Me abolism and Redox Biology, a sec ion o he jou nal F on ie s in Aging Recei ed: 03 Augus 2021 Accep ed: 21 Sep embe 2021 Published: 29 Oc obe 2021 Ci a ion: Hi onen OP, Ky öläinen H, Leh i M and Kainulainen H (2021) Randomized T ial: D-Glyce ic Acid Ac i a es Mi ochond ial Me abolism in 50–60- Yea -Old Heal hy Humans. F on . Aging 2:752636. doi: 10.3389/ agi.2021.752636 Abb e ia ion: ADME, abso p ion, dis ibu ion, me abolism, and exc e ion; ALT, alanine amino ans e ase; AST, aspa a e amino ans e ase; ATP, adenosine iphospha e; bHB, be a-hyd oxybu y ic acid; CK, c ea ine kinase; DGA, D-glyce ic acid; FA oxid, a y acid oxida ion; FAs, a y acids; FATP, a y acid anspo p o ein; FFAs, ee a y acids (To al FAs–TGs); GlycA, glycop o ein ace yls; GLUT, glucose anspo e ; IL-6, in e leukin 6; JyU, Uni e si y o Jy äskylä; LDH, lac a e dehyd ogenase; MCT, monoca boxylic acid anspo e ; NADH, educed nico inamide adenosine dinucleo ide; NAD+, oxidized o m o NADH; OAA, oxaloace a e; OXPHOS, oxida i e phospho yla ion; PC, py u a e ca boxylase; PDH, py u a e dehyd ogenase; PEP, phosphoenolpy u a e Enzymes; PK, Py u a e kinase; TCA, ica boxylic acid cycle; TGs, iglyce ides. F on ie s in Aging | www. on ie sin.o g Oc obe 2021 | Volume 2 | A icle 7526361 ORIGINAL RESEARCH published: 29 Oc obe 2021 doi: 10.3389/ agi.2021.752636 HIGHLIGHTS O al D-glyce ic acid (DGA) adminis a ion causes bo h as and las ing posi i e me abolic e ec s in heal hy 50–60-yea -old humans. O e all mi ochond ial me abolism was ac i a ed by he inc ease in in acellula DGA concen a ion. Bo h he OXPHOS and NADH using anabolic eac ions we e up egula ed in he whole-body and pa icula ly in immune cells and hepa ocy es. Plasma lac a e was s ongly and s a is ically ex emely significan ly educed. Subclinical inflamma ion measu ed by 3 independen me hods was educed bo h in he 4- and 21-days ea men s. Cellula memb ane in eg i y seemed o be sha ply imp o ed. C ea ine kinase and aspa a e amino ans e ase elease o plasma was lowe ed sha ply and s a is ically e y significan ly al eady in 4- days. INTRODUCTION D-glyce ic acid (DGA) is a na u al o ganic acid p esen in e y small amoun s in e eb a es and plan s. Ne e heless, he e a e only a ew scien ific s udies on his small me aboli e. Due o i s small size and low, a ying, concen a ions e en he measu emen o exac DGA concen a ion om fluids, and issues a physiological le els is somewha challenging (Ho mann e al., 1989). In 27–39 yea -old adul s, he DGA concen a ion in plasma was on a e age only 0.3% o he lac a e concen a ion (Ho mann e al., 1993). As a small monoca boxylic acid, DGA molecules dis ibu e h ough monoca boxyla e anspo e s om blood ci cula ion in o issues. In e es ingly in pedia ic pa ien s, he a io o DGA concen a ion in ce eb ospinal fluid o plasma seems o be clea ly highe han ha o o ganic acids in gene al (Ho mann e al., 1993). This may indica e ha he di usion o DGA h ough he blood-b ain ba ie as well as o he memb anes is no o ally fluen . The main enzyme ha me abolizes DGA in humans and animals is glyce a e kinase (GLYCTK).Glyoxyla e educ ase hyd oxypy u a e educ ase (GRHPR) can also oxidize DGA. GLYCTK and GRHPR a e widely exp essed and ac i e in all issues (www.p o eina las.o g/). GLYCTK enzymes possess se e al splice a ian s ha a e localized in cy osol and in mi ochond ia (Guo e al., 2006). Acco ding o Unip o Knowledgebase, 4 splice a ian s a e loca ed in mi ochond ia and 3 in cy osol. GLYCTK kinase eac ion consumes one ATP and simul aneously phospho yla es DGA in o glycoly ic in e media e 2-phosphoglyce a e. The e is a a e inbo n e o called D-glyce ic Acidu ia ha likely ela es o some deficiency in GLYCTK (Sass e al., 2010). Cellula syn hesis o DGA occu s mainly om D-glyce aldehyde (D-GALD) ia aldehyde dehyd ogenase enzymes. These enzymes a e o en mi ochond ially loca ed (S agos e al., 2010). D-GALD is a p oduc o uc ose ca abolism, which occu s mainly in he li e and o a lesse ex en in he in es ines. In e es ingly, skele al muscles and many o he o gans possess GLUT anspo e s ha a e specified in o uc ose in ake om plasma, indica ing clea ly ha D-GALD p oducing uc ose ca abolism also occu s o some ex en in skele al muscle (Zie a h e al., 1995;Scheepe s GRAPHICAL ABSTRACT | DGA ac i a ion ma e ializes in all ac i e issues like muscles, immune sys em, and he li e . F on ie s in Aging | www. on ie sin.o g Oc obe 2021 | Volume 2 | A icle 7526362 Hi onen e al. D-Glyce ic Acid Ac i a es Mi ochond ial Me abolism e al., 2005). In no mal physiological condi ions, D-GALD is phospho yla ed in he cy osol by iose kinase and i en e s glycolysis. In ea lie s udies, he e ec s o DGA adminis a ion ha e been linked o e hanol oxida ion (E iksson e al., 2007;Habe e al., 2011). Oxida ion o e hanol was shown o be accele a ed by a subs an ial 25% in male a s when acu e 500 mg/kg o 100 mg/kg DGA dose was gi en in ape i oneally 1 hou be o e he e hanol dose (E iksson e al., 2007). In he same a icle (E iksson e al., 2007), a 3-weeks non-acu e DGA adminis a ion expe imen wi hin he chow was also epo ed (doses 0, 100, 500, o 1,000 mg/kg o DGA). In his 3-weeks non-acu e DGA adminis a ion expe imen e hanol oxida ion was su p isingly no inc eased. A consis en explana ion o hese appa en ly con adic ing esul s was likely a whole-body ac i a ion o (mi ochond ial) ene gy me abolism a e he acu e DGA dose in he s udied a s. I inc eased ene gy me abolic subs a e demand by he pe iphe al issues om he li e ha caused a mild ene gy dep i a ion in he hepa ocy es 1 h a e he acu e DGA dose. Accele a ed e hanol oxida ion eplenished hepa icene gyhomeos asis(NADH/NAD + - a io) in he DGA ea ed a s. Habe e al. (Habe e al., 2011) s udied DGA in gas ic cells in i o a e 2—3% e hanol-dosed medium. In hei expe imen s, cell iabili y inc eased in 72 h by ce ain doses o DGA, LGA, o acemic DL-GA. The aim o he p esen s udy was o find ou di ec and indi ec indica ions o he ac i a ion o mi ochond ial me abolism by he use o DGA. When easible, he issues a ec ed, and he iming o he esponse we e also e alua ed. Simul aneously, we wan ed o in es iga e possible signs o a mo e pe manen mi ochond ial ac i a ion such as happens a e sus ained adap a ion o a mo e ac i e physical li es yle. Tha kind o p o-ene ge ic e ec may lead o posi i e changes in heal h isk ac o s such as de e io a ed cell memb anes in eg i y and ele a ed sys emic inflamma ion (Kim e al., 2019;To as e al., 2020;Dias and Nylands ed, 2021). MATERIALS AND METHODS Al oge he 30 heal hy pa icipan s aged 50–60 yea s we e ca e ully selec ed ou o 45 heal hy and sui able olun ee s o o m he p esen s udy g oup. The olun ee s we e ec ui ed h ough 600 le e s sen andomly o age-ma ched men and women om he a ea o Cen al Finland. All he pa icipan s we e in o med o he expe imen al design, and he benefi s and possible isks ha could be associa ed wi h he s udy p io o signing an in o med consen o olun a ily pa icipa e in he s udy. All s udies we e conduc ed in line wi h he s a emen o he E hical commi ee o he Cen al Finland Heal h Ca e Dis ic (Dn o 1U/2019, KSSHP). Cha ac e is ics o he S udy G oup The age g oup o 50–60 yea s was chosen because a ha age sys emic inflamma ion ma ke s a e on a e age somewha ele a ed e en in appa en ly heal hy pe sons (Wyczalkowska-Tomasik e al., 2016). BMI o he pa icipan s was es ic ed o 18.5–32.0 so ha he e could no be clea ly unde - o o e weigh pa icipan s. All pa icipan s we e Caucasians (Table 1). Any his o y o ca dio ascula diseases was an exclusion c i e ion. Also, hose pe sons who had o a el ex ensi ely du ing he s udy we e excluded. No mal and s able beha io du ing he s udy was enhanced by pe sonal dia ies and eminde emails. Mo ning in e iews we e ca ied ou indi idually when pa icipan s a i ed a he s udy si e wi hin a minimum o 30 min be o e he fi s blood sample. Also, he heal h s a us o he pa icipan , compa able ci cums ances, and he iming o he las dose we e always checked. All he pa icipan s who came o he Day 0 measu emen s comple ed he whole s udy. Ne e heless, h ee o he selec ed pe sons canceled a he las minu e be o e he Day 0 measu emen due o mild flu o simila symp oms. S udy Se up and Measu emen s The es se up was double-blinded. Measu emen s we e always pe o med on he same weekday (F iday o Sa u day) o each pa icipan . To achie e mo e compa able same-weekday measu emen s, we added wo eco e y days a e he Day0 VO 2 max measu emen s (Figu e 1A). Fo simplici y, we call he second measu emen day he “Day4”because i was aken a e he 4 days o DGA egimen e en hough he ac ual day was he 7 h. The placebo g oup was chosen andomly among women and men sepa a ely be o ehand. The exis ence o he placebo g oup h oughou he s udy was in o med o he pa icipan s. In p ac ice, he numbe o placebo- ea ed pa icipan s was ze o un il he Day4 measu emen s. F om ha day onwa ds, he numbe o pa icipan s in he placebo g oup was inc eased o 10, i.e., he placebo g oup exis ed de ac o only o he 2-weeks ollow-up pe iod (Figu es 1A,B). All mo ning blood samples we e aken 12 h a e he las DGA o placebo dose, i.e., non-acu ely. Blood samples o each pa icipan we e always aken a he same ime in he mo ning (+/−2min). Addi ionally, h oughou he whole s udy pe iod ully no mal bu s able li ing condi ions we e equi ed. 1 Tes Subs ances and Doses, P epa a ions, and Adminis a ion D-glyce ic acid calcium sal (DGAcs) and placebo (E509/calcium chlo ide) we e dissol ed in o 1.5 L bo les o wa e be o ehand o each pa icipan . The calcula ed dose o DGA o placebo was o be d unk in he mo ning and he e ening. In he placebo g oup, he e was an equal mola amoun o calcium wi h wa e . DGA was TABLE 1 | Cha ac e is ics o he s udy g oup. Day0 me ics Main s udy g oup DGA g oup Placebo g oup A e age Age 56 yea s ( om 50.3 o 60.9) 56.5 55.2 A e age BMI 25.3 ( om 20.1 o 31.7) 25.0 25.8 A g. VO 2 max 35.5 ( om 21.8 o 48.8) 35.1 36.1 Female/Male 16 emales and 11 males 10/7 6/4 No es: uni in BMI weigh in kg/(leng h in me e s) 2 , uni in VO 2 max O 2 ml/kg/min. VO 2 max es was based on indi ec measu emen wi h a bicycle e gome e (San ila e al., 2013). All pa icipan s execu ed his ou ine es o he JyU Spo s Labo a o y success ully. 1 One pe son had o be ully excluded om he final esul s because o 10 days p io he had ini ia ed a low-calo ie die . Ano he pe son was excluded om he Day4 esul s because o epo ed significan wo k s ess he p e ious nigh . F on ie s in Aging | www. on ie sin.o g Oc obe 2021 | Volume 2 | A icle 7526363 Hi onen e al. D-Glyce ic Acid Ac i a es Mi ochond ial Me abolism p oduced and pu ified in o calcium sal a a molecula le el by VTT Technical Resea ch Cen e o Finland (VTT) and Replicon Heal h Oy in collabo a ion (Habe e al., 2009). On op o VTT, he pu i y o he ba ch was es ed by he Finnish Food Au ho i y ( esidues and concen a ion), and Pha ma o y Oy o enan iome s. Be o e his s udy, pa o he ba ch was used in ex ensi e comme cial ea ing expe imen s. In he hepa ocy e s udy (Supplemen a y P esen a ion 3), used DGAcs we e om Sigma-Ald ich (3,67,494). Selec ed doses, egimens, and measu emen imings in he p esen s udy we e based on ea lie in i o and in i o p e- es s ela ed o he egula o y accep ance p ocesses o DGA. The e ec i e dose o DGA o he fi s 4-days was 3.33 mg pe kg body weigh wice pe day. Tha dose was p e- es ed in small-scale human pilo ing as a na u al ood addi i e (Hi onen e al., 2015). In he 14-days ollow-up pe iod he DGA egimen was educed o hal because we wan ed o explo e he smalles su ficien dose o humans. (As a compa ison, he highes DGA dose pe kg body weigh (BW) in he 3-weeks a expe imen (E iksson e al., 2007) was mo e han 200- imes he dose used in he cu en human s udy du ing he ollow-up pe iod. As epo ed in he a icle (E iksson e al., 2007)“no oxici y”was obse ed in s udied a s a any o he doses.) Blood Samples Blood samples we e d awn om he an ecubi al ein o each pa icipan , always a he same ime in he mo ning. The samples we e immedia ely cooled and cen i uged in hepa in plasma ubes and s o ed in 2 ml po ions a −80°C. Plasma samples we e analyzed in Nigh ingale Heal h Oy (Nuclea Magne ic Resonance (NMR) echnology wi h egula o y app o al o diagnos ics) (Soininen e al., 2015), excep o CK, AST, and ALT ha we e analyzed in Synlab Finland wi h clinically acc edi ed s anda d me hods. Insulin, hsCRP, and IL-6 we e measu ed a he JyU labo a o y using alida ed ki s, and plasma DGA concen a ion in VTT using gas ch oma og aphy-mass spec ome y echnique. RNA Sequencing mRNA exp essions we e measu ed om whi e blood cells (WBCs). WBCs samples we e aken a he same ime as mo ning as ing and es ing blood samples. Whole-genome mRNA sequencing was conduc ed by he Ins i u e o Molecula Medicine Genomics Uni (FIMM/Uni e si y o Helsinki) (Cos ello e al., 2018). Collec ion o whole blood samples in o PAXgene Blood RNA Tubes and ex ac ion o mRNA was conduc ed acco ding o manu ac u e ’s ins uc ions. Human in i o Side S udy Wi h P ima y Hepa ocy es In ou pilo s udies wi h human p ima y hepa ocy es and a p ima y co ical neu ons, i was no iced ha ene gy consump ion seemed o be ac i a ed in he DGA ea ed cells compa ed o 0-con ols (Hi onen e al., 2015). To find ou how ene gy consump ion was inc eased, we measu ed NAD+/NADH - a io om he hepa ocy es o h ee human FIGURE 1 | Ou line o he S udy: Timelines and measu emen s (A), main phases o s udy (B), and he acu e measu emen s (C). F on ie s in Aging | www. on ie sin.o g Oc obe 2021 | Volume 2 | A icle 7526364 Hi onen e al. D-Glyce ic Acid Ac i a es Mi ochond ial Me abolism dono s. The esul s a e scien ifically epo ed o he fi s ime in Supplemen a y P esen a ion 3. S a is ical Tes s and Su ficien Numbe o Obse a ions Each pe son was used as hei own con ol. Tha way indi idual “noise ac o s”could be elimina ed and we we e able o pai wise es whe he he in aindi idual esponses we e simila among s udy pe sons. Fu he mo e, o achie e s a is ically unambiguous esul s all blinded pa icipan s we e in he same compa ison g oup du ing he fi s week. P e-assessmen on he su ficien g oup size was based on he esul s om ou pilo es s and ela ed ola ili ies. Among ou ela i ely homogenous s udy g oup wi h ully compa able in aindi idual measu emen poin s, he selec ed g oup size u ned ou o be clea ly su ficien . S a is ical es s we e conduc ed using IBM SPSS s a is ics so wa e and Mic oso Excel. P esen ed s a is ical es esul s a e mos ly om pa ame ic S uden ’s - es s. When N<15, we checked he no mali y (Rou ledge, 2020) o he unde lying da a using he Kolmogo o -Smi no es . Non-pa ame ic Mann- Whi ney U- es o SIGN es s we e used when clea ly needed. p- alue <0.05, <0.01, and <0.001 in a one-sided - es we e conside ed s a is ically significan , e y significan and ex emely significan espec i ely. All p esen ed es s we e p ede e mined o de i ed om p ede e mined es se ings. FIGURE 2 | Rep esen a i e ene gy subs a es unde he 4-days DGA egimen, 12 h om las DGA dose, and DGA and insulin (A). Schema ic flows o all epo ed plasma ene gy subs a es a whole-body le el (B). S a is ical es s a e based on in a-indi idual changes (pai ed - es s). E o ba s a e s anda d e o s o he mean (SEM) o he indi idual changes. F on ie s in Aging | www. on ie sin.o g Oc obe 2021 | Volume 2 | A icle 7526365 Hi onen e al. D-Glyce ic Acid Ac i a es Mi ochond ial Me abolism RESULTS AND DISCUSSION The 4- and 21-days changes o ca e ully selec ed plasma me ics and mRNA exp essions om WBCs we e used as indi ec p ima y ma ke s o he ac i a ion o mi ochond ial me abolism. Al eady a e 4 days o he DGA egimen, he e is a significan down egula ion o all plasma ene gy subs a es (Figu e 2). Especially he s ong decline in plasma lac a e indica es an up egula ion o he whole-body mi ochond ial (ene gy) me abolism (Figu e 3). In sec ion @ADME and he Timing o he Response o he DGA Ac i a ion@, we sho ly ou line he ADME o DGA and how he esponse o he DGA egimen ma e ializes om he acu e single-dose owa ds he 4 days adminis a ion. The 21-days esul s show ha he whole-body homeos asis has been es o ed bu a a mo e “ene ge ic”le el, he li e and muscle unc ions ha ing especially imp o ed (Figu e 4). Hepa ic enhancemen is isible om AST and ALT esul s and muscula imp o emen s om CK esul s (Figu e 5). Low-g ade inflamma o y ma ke s IL-6 and GlycA we e also ou ex-an e- defined p ima y ma ke s. Posi i e esul s on he educ ion o ch onic inflamma ion a e p esen ed in Figu e 5 and ela ed analyses wi h hsCRP as an addi ional p oo o concep . No ably, CK, AST, GlycA, and hsCRP showed a s ong posi i e esponse al eady o he 4-days DGA egimen (Figu es 5A,B,D,E). Day 0 s a ing alues and he 4 days %-changes o all used plasma me ics a e anked by indi idual VO 2 max esul s and p esen ed in Supplemen a y P esen a ion 1. Cellula le el s udies on WBCs and hepa ocy es a e epo ed in Supplemen a y P esen a ion 2, 3 espec i ely. Acu e 45-min measu emen s on IL-6 and insulin a e p esen ed a he end o Supplemen a y P esen a ion 1 (“VO 2 max co ela ing ma ke s2”) and in Supplemen a y P esen a ion 4. Re e ences a e made o supplemen s when needed o suppo conclusions. Plasma Ene gy Subs a e Responses o he 4-Days DGA Regimen Plasma ene gy subs a e concen a ions eflec in acellula (cy osolic) concen a ions ia plasma memb ane anspo e s (Figu e 3). Main ene gy subs a es like glucose, TGs, and FAs can be es uc u ed and s o ed in cells and hus a e no exc e ed back in o he plasma. Smalle cy osolic me aboli es like lac a e and py u a e can be e ficien ly impo ed o expo ed ia plasma memb ane monoca boxylic acid anspo e s (Figu e 3). FIGURE 3 | Lac a e, py u a e, glucose, and TGs/FAs inflow om plasma and hei in acellula me abolism in a cell ha can use bo h FAs and glucose as he main sou ce o ene gy. Independen ly o hei final use in me abolism (li e o pe iphe al issues), he in acellula di ec ion o lac a e ia py u a e is owa ds mi ochond ia. This is because he cy osolic PK eac ion is i e e sible. TCA p oduces mos o he NADH o OXPHOS. Glycolysis p oduces 2 NADH pe one glucose. The e exis ed a e y s ong co ela ion be ween he changes o plasma py u a e and lac a e independen ly o placebo o DGA ea men (>0.90). This co ela ion s ongly indica es ha MCTs a e able o balance plasma memb ane concen a ion di e ences e ficien ly. F on ie s in Aging | www. on ie sin.o g Oc obe 2021 | Volume 2 | A icle 7526366 Hi onen e al. D-Glyce ic Acid Ac i a es Mi ochond ial Me abolism P esen ed subs a es in Figu e 2A ep esen all main nu i ional ca ego ies i.e., a s, ca bohyd a es, amino acids, ke one bodies, and ca boxylic acids. They co e some 90% o used ene gy subs a es a es . All plasma concen a ions o hese ene gy subs a es we e down egula ed by he 4 days DGA egimen (Figu e 2A). Obse ed consis en esponse ex emely likely eflec ed a dec ease in in acellula concen a ions o he subs a es because he o he possibili y o inc eased exc e ion ou o he body om plasma due o he DGA egimen can be su ficien ly uled ou by ca e ul o gan and subs a e-le el assessmen s (Figu es 2A,B). In acellula ene gy subs a e concen a ions may dec ease h ough me abolism in o ei he he TCA (Ma ínez-Reyes and Chandel, 2020), in o anabolic eac ions, o in o in acellula ene gy s o es. All hese h ee me abolic al e na i es ela e somehow o mi ochond ial me abolism because mos o he anabolic eac ions like gluco- and glyce oneogenesis ma e ialize ia mi ochond ia (Figu e 3). Also, FA oxida ion ma e ializes in mi ochond ia. Ene gy Subs a e Ne Inflow In o Cells Poin o Mi ochond ial Ac i a ion and Inc eased NADH/NAD + -Ra io We ound ha he 4-days DGA egimen caused an ex emely significan 21% a e age decline in plasma lac a e (p0.0009, Figu e 2A). The e does no seem o be any de ia ing pe sons in lac a e esponses despi e he wide s a ing ange o lac a e concen a ions (Supplemen a y P esen a ion 1, Figu e A). When lac a e is impo ed o he cells, i s in acellula me abolic ou eisalways iaLDHandpy u a e owa dsmi ochond ia (Figu e 3). In ac , all p esen ed ene gy subs a es e en ually end in mi ochond ial me abolism (when oxygen is p esen ). Thus, he esul s p esen ed in Figu e 2A we e a s ong indica ion o inc eased mi ochond ial me abolism a he whole-body le el. Fu he mo e, he final ca abolism o all ene gy subs a es in o CO 2 p oduces ene gy-con aining NADH molecules. Mos o hem a e p oduced by heTCAandusedby heOXPHOS(Figu e 3). All in all, ou esul s show an inc ease in o e all NADH (ene gy) le el in cells ia DGA ac i a ion. We pe o med an addi ional in i o expe imen wi h cul u ed hepa ocy es. Indeed, NADH gene a ion was significan ly ac i a ed 3 h a e he las DGA dose (Supplemen a y P esen a ion 3). The 4-days DGA Regimen Seems o Cause Tempo a y Hepa ic Lac a e Sho age The fi s ou ba s in Figu e 2A ep esen ene gy subs a es ha a e mos ly p o ided by he main aining o gans (a ows in Figu e 2B) and he las 5 ba s (excl. he DGA and insulin) ep esen subs a es ha a e mos ly impo ed o he li e o gluco- and glyce oneogenesis (a ows in Figu e 2B). The e is an in e es ing di e ence be ween he fi s 4 and nex 5 ba s in Figu e 2B. Al hough all a e down, he subs a es going owa ds he li e a e all s a is ically significan ly o e en ex emely significan ly down egula ed, bu none o he educ ions in he subs a es going owa ds pe iphe al issues is significan . I seems ha du ing he 4 days DGA ac i a ion a sca ci y o gluco- and glyce oneogenic plasma subs a es owa ds he li e had clea ly de eloped. Fu he mo e, om he olume dec eases ( igh -hand scale, ho izon al ba s in Figu e 2A)wecan clea ly obse e ha he sca ci y was almos ully due o a decline in lac a e. A s a is ically e y significan 21% decline in plasma glyce ol (Figu e 2A) demons a es lac a e sho age o hepa ic glyce oneogenesis. Glyce ol is no a di ec ene gy subs a e and hus he DGA egimen should no ha e any majo e ec on i . Addi ionally, glyce ol kinase enzymes a e ac i e mos ly in he li e . A obus 21% decline in glyce ol indica es ha he li e s a ed o impo ee glyce ol om plasma o o m glyce ol phospha e by hepa ic glyce ol kinase eac ions and o husly compensa e he sha p educ ion o plasma lac a e. Lac a e is he main subs a e o hepa ic FIGURE 4 | 21 days (A) and 14 days (B) %-changes in selec ed plasma ene gy me aboli es and insulin. S a is ical es s a e based on in a-indi idual changes. In Figu e 4A he changes a e om Day 0 and in Figu e 4B om Day 4. Ho izon al ba s in bHB and Insulin indica e s a is ically significan de ia ion be ween he placebo and DGA subg oups. F on ie s in Aging | www. on ie sin.o g Oc obe 2021 | Volume 2 | A icle 7526367 Hi onen e al. D-Glyce ic Acid Ac i a es Mi ochond ial Me abolism glyce oneogenesis and glyce ol phospha e is i s p oduc (Nye e al., 2008). Fu he p oo comes om he su p ising bu empo a y decline in plasma glucose, Figu e 2A which also poin s o a mild sho age o lac a e. This ime o hepa ic gluconeogenesis a Day 4 (Figu e 2A). Insulin Hinde s Glucose In ake o Tissues Likely Because o Inc eased In acellula Glucose S o age due o he DGA Ac i a ion A he whole-body le el, he empo a y and benign lac a e sho age has likely de eloped due o inc eased glycogen (glucose) and TGs/FAs s o age in o pe iphe al issues like skele al muscles. Plasma glucose le el declines s a is ically almos significan ly despi e less s imula ion om insulin (Figu e 2A). One c edible explana ion in es ing measu emen s was ha on op o he lac a e sho age also glycogen s o age in pe iphe al issues was likely o ha e been inc eased, as also happens a e physical exe cise by skele al muscles (He mansen e al., 1970;Goodwin, 2010). Also, TGs and FFAs seemed o be used/s o ed a a sligh ly highe a e unde he DGA egimen (Figu es 2A,4A)(Ha g ea es and Sp ie , 2020). Mo e de ailed analysis on plasma TGs and FFAs esponse o he 4-days DGA egimen is p esen ed in Supplemen a y P esen a ion 1 (Figu es K,M, and K and M clus e ed). The 4-Days DGA Regimen Causes S ong Modula ion o Mi ochond ia-Rela ed mRNA Exp essions in he Immune Cells The mRNA exp essions o ce ain ATP syn hase genes we e s ongly down egula ed a Day4 (Supplemen a y P esen a ion 2,Supplemen a y P esen a ion 1, Figu es B,C). Cellula ATP p oduc ion is igh ly egula ed o a oid excessi e ATP p oduc ion. Also, he mRNA exp ession o he PC gene (Figu e 3)was up egula ed s a is ically e y significan ly du ing he 4 days DGA egimen (Supplemen a y P esen a ion 2, Figu e B). In mi ochond ia he PC enzyme di ec s incoming ene gy subs a e FIGURE 5 | Enzyme elease o c ea ine kinase (A), aspa a e amino ans e ase (B), and alanine amino ans e ase (C) in o he plasma, and low-g ade inflamma ion ma ke s GlycA (D), hsCRP (E), and IL-6 (F). All s a is ical es s a e based on in a-indi idual changes. SEM e o ba s a e calcula ed om absolu e alues. No es: 1) Day 21 ba s in (A–F) a e indexed o Day 0 ba s so ha he Day 21 esul s ully eflec he changes om he ele an DGA and placebo subg oups a Day 0. 2) The e was one clea ou lie in he 4-days esul in CK and AST om he same pa icipan (see Supplemen a y P esen a ion 1, Figu es H,I), and one ou lie in IL-6 Day 0 esul s (Supplemen a y P esen a ion 1, Figu e P). Addi ionally, in GlycA (Figu e 3D) he highes 1/3 anked by VO 2 max de ia ed clea ly om he o he pa icipan s and om hsCRP (see Supplemen a y P esen a ion 1, Figu es N,O). These obse a ions ha e been excluded o achie e no mali y o he da a. Impo an ly, e en including hese ou lie s, he espec i e changes in CK, AST, GlycA, and IL-6 would ha e been s a is ically significan . 3) The hsCRP da a is non-no mal due o e y high ola ili y. Due o he high ola ili y median alues o hsCRP a e p esen ed in (E) and a non-pa ame ic Sign es was used o he 4-days change. 4) All o he ba s ep esen a i hme ic means and he in aindi idual changes o all o he s we e no mally dis ibu ed. S a is ically mo e powe ul pai ed - es was used when compa ing he in aindi idual changes om Day 0 in (A,B,D, and F). 5) Na u ally, when compa ing he changes om Day 0 o Day 21 in he placebo g oup s. he DGA g oup (ALT and IL-6, (C,F)), a pai ed - es could no be used. Ins ead, a non-pai ed - es was used o compa e he changes in he placebo s. DGA o ALT and IL-6. F on ie s in Aging | www. on ie sin.o g Oc obe 2021 | Volume 2 | A icle 7526368 Hi onen e al. D-Glyce ic Acid Ac i a es Mi ochond ial Me abolism