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

This is a self-archived version of an original article. This version may differ from the original in pagination and typographic details. Author(s): Title: Year: Version: Copyright: Rights: Rights url: Please cite the original version: CC BY 4.0 https://creativecommons.org/licenses/by/4.0/ Randomized Trial : D-Glyceric Acid Activates Mitochondrial Metabolism in 50–60-YearOld Healthy Humans © 2021 Hirvonen, Kyröläinen, Lehti and Kainulainen. Published version Hirvonen, O. Petteri; Kyröläinen, Heikki; Lehti, Maarit; Kainulainen, Heikki Hirvonen, O. P., Kyröläinen, H., Lehti, M., & Kainulainen, H. (2021). Randomized Trial : D-Glyceric Acid Activates Mitochondrial Metabolism in 50–60-Year-Old Healthy Humans. Frontiers in Aging, 2, Article 752636. https://doi.org/10.3389/fragi.2021.752636 2021 Randomized Trial: D-Glyceric Acid Activates Mitochondrial Metabolism in 50–60-Year-Old Healthy Humans O. Petteri Hirvonen*, Heikki Kyröläinen, Maarit Lehti and Heikki Kainulainen Faculty of Sport and Health Sciences, Neuromuscular Research Center, University of Jyväskylä, Jyväskylä, Finland Background: Based on earlier studies, natural metabolite D-glyceric acid (DGA) does not seem to play any role in whole-body metabolism. Nevertheless, one ethanol oxidationrelated rat study with controversial results raised our interest. According to preparatory studies for the regulatory approval of DGA, some highly conserved mechanism seems to subtly activate the cellular energy metabolism. Therefore, the present 25-days doubleblind human study with placebo control was initiated. Purpose: The main target in the present study with 27 healthy 50–60-year-old human volunteers was to find out whether an “acute”4-days and a longer 21-days exogenous DGA regimen caused moderate activation of the mitochondrial energy metabolism. The simultaneous target was to find out whether a halved dose of DGA continued to be an effective regimen. Main Findings: The results revealed the following statistically significant findings: 1) plasma concentrations of metabolites related to aerobic energy production, especially lactate, were strongly reduced, 2) systemic inflammation was lowered both in 4and 21days, 3) mitochondria-related mRNA expressions in circulating immune cells were noticeably modulated at Day4, 4) cellular membrane integrity seemed to be sharply enhanced, and 5) cellular NADH/NAD + -ratio was upregulated. Conclusion: Mitochondrial metabolism was clearly upregulated at the whole-body level in both 4and 21 days. At the same time, the effect of DGA was very well tolerated. Based on received solid results, the DGA regimen may alleviate acute and chronic energy metabolic challenges in main organs like the liver, CNS, and skeletal muscles. Enhanced membrane integrity combined with lower systemic inflammation and activated metabolic flows by the DGA regimen may be beneficial especially for the aging population. Keywords: mitochondrial activation, re-oxidation, membrane integrity, subclinical inflammation, DGA activation Edited by: Smijin K Soman, University of Nevada, United States Reviewed by: Ting Liu, Johns Hopkins University, United States Julie Reisz Haines, University of Colorado Denver, United States *Correspondence: O. Petteri Hirvonen [email protected].fi Specialty section: This article was submitted to Aging, Metabolism and Redox Biology, a section of the journal Frontiers in Aging Received: 03 August 2021 Accepted: 21 September 2021 Published: 29 October 2021 Citation: Hirvonen OP, Kyröläinen H, Lehti M and Kainulainen H (2021) Randomized Trial: D-Glyceric Acid Activates Mitochondrial Metabolism in 50–60Year-Old Healthy Humans. Front. Aging 2:752636. doi: 10.3389/fragi.2021.752636 Abbreviation: ADME, absorption, distribution, metabolism, and excretion; ALT, alanine aminotransferase; AST, aspartate aminotransferase; ATP, adenosine triphosphate; bHB, beta-hydroxybutyric acid; CK, creatine kinase; DGA, D-glyceric acid; FA oxid, fatty acid oxidation; FAs, fatty acids; FATP, fatty acid transport protein; FFAs, free fatty acids (Total FAs–TGs); GlycA, glycoprotein acetyls; GLUT, glucose transporter; IL-6, interleukin 6; JyU, University of Jyväskylä; LDH, lactate dehydrogenase; MCT, monocarboxylic acid transporter; NADH, reduced nicotinamide adenosine dinucleotide; NAD+, oxidized form of NADH; OAA, oxaloacetate; OXPHOS, oxidative phosphorylation; PC, pyruvate carboxylase; PDH, pyruvate dehydrogenase; PEP, phosphoenolpyruvate Enzymes; PK, Pyruvate kinase; TCA, tricarboxylic acid cycle; TGs, triglycerides. Frontiers in Aging | www.frontiersin.org October 2021 | Volume 2 | Article 7526361 ORIGINAL RESEARCH published: 29 October 2021 doi: 10.3389/fragi.2021.752636 HIGHLIGHTS Oral D-glyceric acid (DGA) administration causes both fast and lasting positive metabolic effects in healthy 50–60-year-old humans. Overall mitochondrial metabolism was activated by the increase in intracellular DGA concentration. Both the OXPHOS and NADH using anabolic reactions were upregulated in the whole-body and particularly in immune cells and hepatocytes. Plasma lactate was strongly and statistically extremely significantly reduced. Subclinical inflammation measured by 3 independent methods was reduced both in the 4and 21-days treatments. Cellular membrane integrity seemed to be sharply improved. Creatine kinase and aspartate aminotransferase release to plasma was lowered sharply and statistically very significantly already in 4days. INTRODUCTION D-glyceric acid (DGA) is a natural organic acid present in very small amounts in vertebrates and plants. Nevertheless, there are only a few scientific studies on this small metabolite. Due to its small size and low, varying, concentrations even the measurement of exact DGA concentration from fluids, and tissues at physiological levels is somewhat challenging (Hoffmann et al., 1989). In 27–39 year-old adults, the DGA concentration in plasma was on average only 0.3% of the lactate concentration (Hoffmann et al., 1993). As a small monocarboxylic acid, DGA molecules distribute through monocarboxylate transporters from blood circulation into tissues. Interestingly in pediatric patients, the ratio of DGA concentration in cerebrospinal fluid to plasma seems to be clearly higher than that of organic acids in general (Hoffmann et al., 1993). This may indicate that the diffusion of DGA through the blood-brain barrier as well as other membranes is not totally fluent. The main enzyme that metabolizes DGA in humans and animals is glycerate kinase (GLYCTK).Glyoxylate reductase hydroxypyruvate reductase (GRHPR) can also oxidize DGA. GLYCTK and GRHPR are widely expressed and active in all tissues (www.proteinatlas.org/). GLYCTK enzymes possess several splice variants that are localized in cytosol and in mitochondria (Guo et al., 2006). According to Uniprot Knowledgebase, 4 splice variants are located in mitochondria and 3 in cytosol. GLYCTK kinase reaction consumes one ATP and simultaneously phosphorylates DGA into glycolytic intermediate 2-phosphoglycerate. There is a rare inborn error called D-glyceric Aciduria that likely relates to some deficiency in GLYCTK (Sass et al., 2010). Cellular synthesis of DGA occurs mainly from D-glyceraldehyde (D-GALD) via aldehyde dehydrogenase enzymes. These enzymes are often mitochondrially located (Stagos et al., 2010). D-GALD is a product of fructose catabolism, which occurs mainly in the liver and to a lesser extent in the intestines. Interestingly, skeletal muscles and many other organs possess GLUT transporters that are specified into fructose intake from plasma, indicating clearly that D-GALD producing fructose catabolism also occurs to some extent in skeletal muscle (Zierath et al., 1995;Scheepers GRAPHICAL ABSTRACT | DGA activation materializes in all active tissues like muscles, immune system, and the liver. Frontiers in Aging | www.frontiersin.org October 2021 | Volume 2 | Article 7526362 Hirvonen et al. D-Glyceric Acid Activates Mitochondrial Metabolism et al., 2005). In normal physiological conditions, D-GALD is phosphorylated in the cytosol by triose kinase and it enters glycolysis. In earlier studies, the effects of DGA administration have been linked to ethanol oxidation (Eriksson et al., 2007;Habe et al., 2011). Oxidation of ethanol was shown to be accelerated by a substantial 25% in male rats when acute 500 mg/kg or 100 mg/kg DGA dose was given intraperitoneally 1 hour before the ethanol dose (Eriksson et al., 2007). In the same article (Eriksson et al., 2007), a 3-weeks non-acute DGA administration experiment within the chow was also reported (doses 0, 100, 500, or 1,000 mg/kg of DGA). In this 3-weeks non-acute DGA administration experiment ethanol oxidation was surprisingly not increased. A consistent explanation for these apparently contradicting results was likely a whole-body activation of (mitochondrial) energy metabolism after the acute DGA dose in the studied rats. It increased energy metabolic substrate demand by the peripheral tissues from the liver that caused a mild energy deprivation in the hepatocytes 1 h after the acute DGA dose. Accelerated ethanol oxidation replenished hepaticenergyhomeostasis(NADH/NAD + -ratio) in the DGA treated rats. Habe et al. (Habe et al., 2011) studied DGA in gastric cells in vitro after 2—3% ethanol-dosed medium. In their experiments, cell viability increased in 72 h by certain doses of DGA, LGA, or racemic DL-GA. The aim of the present study was to find out direct and indirect indications of the activation of mitochondrial metabolism by the use of DGA. When feasible, the tissues affected, and the timing of the response were also evaluated. Simultaneously, we wanted to investigate possible signs of a more permanent mitochondrial activation such as happens after sustained adaptation to a more active physical lifestyle. That kind of pro-energetic effect may lead to positive changes in health risk factors such as deteriorated cell membranes integrity and elevated systemic inflammation (Kim et al., 2019;Tofas et al., 2020;Dias and Nylandsted, 2021). MATERIALS AND METHODS Altogether 30 healthy participants aged 50–60 years were carefully selected out of 45 healthy and suitable volunteers to form the present study group. The volunteers were recruited through 600 letters sent randomly to age-matched men and women from the area of Central Finland. All the participants were informed of the experimental design, and the benefits and possible risks that could be associated with the study prior to signing an informed consent to voluntarily participate in the study. All studies were conducted in line with the statement of the Ethical committee of the Central Finland Health Care District (Dnro 1U/2019, KSSHP). Characteristics of the Study Group The age group of 50–60 years was chosen because at that age systemic inflammation markers are on average somewhat elevated even in apparently healthy persons (Wyczalkowska-Tomasik et al., 2016). BMI of the participants was restricted to 18.5–32.0 so that there could not be clearly underor overweight participants. All participants were Caucasians (Table 1). Any history of cardiovascular diseases was an exclusion criterion. Also, those persons who had to travel extensively during the study were excluded. Normal and stable behavior during the study was enhanced by personal diaries and reminder emails. Morning interviews were carried out individually when participants arrived at the study site within a minimum of 30 min before the first blood sample. Also, the health status of the participant, comparable circumstances, and the timing of the last dose were always checked. All the participants who came to the Day 0 measurements completed the whole study. Nevertheless, three of the selected persons canceled at the last minute before the Day 0 measurement due to mild flu or similar symptoms. Study Setup and Measurements The test setup was double-blinded. Measurements were always performed on the same weekday (Friday or Saturday) for each participant. To achieve more comparable same-weekday measurements, we added two recovery days after the Day0 VO 2 max measurements (Figure 1A). For simplicity, we call the second measurement day the “Day4”because it was taken after the 4 days of DGA regimen even though the actual day was the 7th. The placebo group was chosen randomly among women and men separately beforehand. The existence of the placebo group throughout the study was informed to the participants. In practice, the number of placebo-treated participants was zero until the Day4 measurements. From that day onwards, the number of participants in the placebo group was increased to 10, i.e., the placebo group existed de facto only for the 2-weeks follow-up period (Figures 1A,B). All morning blood samples were taken 12 h after the last DGA or placebo dose, i.e., non-acutely. Blood samples for each participant were always taken at the same time in the morning (+/−2min). Additionally, throughout the whole study period fully normal but stable living conditions were required. 1 Test Substances and Doses, Preparations, and Administration D-glyceric acid calcium salt (DGAcs) and placebo (E509/calcium chloride) were dissolved into 1.5 L bottles of water beforehand for each participant. The calculated dose of DGA or placebo was to be drunk in the morning and the evening. In the placebo group, there was an equal molar amount of calcium with water. DGA was TABLE 1 | Characteristics of the study group. Day0 metrics Main study group DGA group Placebo group Average Age 56 years (from 50.3 to 60.9) 56.5 55.2 Average BMI 25.3 (from 20.1 to 31.7) 25.0 25.8 Avg. VO 2 max 35.5 (from 21.8 to 48.8) 35.1 36.1 Female/Male 16 females and 11 males 10/7 6/4 Notes: unit in BMI weight in kg/(length in meters) 2 , unit in VO 2 max O 2 ml/kg/min. VO 2 max test was based on indirect measurement with a bicycle ergometer (Santtila et al., 2013). All participants executed this routine test of the JyU Sports Laboratory successfully. 1 One person had to be fully excluded from the final results because of 10 days prior he had initiated a low-calorie diet. Another person was excluded from the Day4 results because of reported significant work stress the previous night. Frontiers in Aging | www.frontiersin.org October 2021 | Volume 2 | Article 7526363 Hirvonen et al. D-Glyceric Acid Activates Mitochondrial Metabolism produced and purified into calcium salt at a molecular level by VTT Technical Research Centre of Finland (VTT) and Replicon Health Oy in collaboration (Habe et al., 2009). On top of VTT, the purity of the batch was tested by the Finnish Food Authority (residues and concentration), and Pharmatory Oy for enantiomers. Before this study, part of the batch was used in extensive commercial rearing experiments. In the hepatocyte study (Supplementary Presentation 3), used DGAcs were from Sigma-Aldrich (3,67,494). Selected doses, regimens, and measurement timings in the present study were based on earlier in vitro and in vivo pre-tests related to the regulatory acceptance processes of DGA. The effective dose of DGA for the first 4-days was 3.33 mg per kg body weight twice per day. That dose was pre-tested in small-scale human piloting as a natural food additive (Hirvonen et al., 2015). In the 14-days follow-up period the DGA regimen was reduced to half because we wanted to explore the smallest sufficient dose for humans. (As a comparison, the highest DGA dose per kg body weight (BW) in the 3-weeks rat experiment (Eriksson et al., 2007) was more than 200-times the dose used in the current human study during the follow-up period. As reported in the article (Eriksson et al., 2007)“no toxicity”was observed in studied rats at any of the doses.) Blood Samples Blood samples were drawn from the antecubital vein of each participant, always at the same time in the morning. The samples were immediately cooled and centrifuged in heparin plasma tubes and stored in 2 ml portions at −80°C. Plasma samples were analyzed in Nightingale Health Oy (Nuclear Magnetic Resonance (NMR) technology with regulatory approval for diagnostics) (Soininen et al., 2015), except for CK, AST, and ALT that were analyzed in Synlab Finland with clinically accredited standard methods. Insulin, hsCRP, and IL-6 were measured at the JyU laboratory using validated kits, and plasma DGA concentration in VTT using gas chromatography-mass spectrometry technique. RNA Sequencing mRNA expressions were measured from white blood cells (WBCs). WBCs samples were taken at the same time as morning fasting and resting blood samples. Whole-genome mRNA sequencing was conducted by the Institute for Molecular Medicine Genomics Unit (FIMM/University of Helsinki) (Costello et al., 2018). Collection of whole blood samples into PAXgene Blood RNA Tubes and extraction of mRNA was conducted according to manufacturer’s instructions. Human in vitro Side Study With Primary Hepatocytes In our pilot studies with human primary hepatocytes and rat primary cortical neurons, it was noticed that energy consumption seemed to be activated in the DGA treated cells compared to 0-controls (Hirvonen et al., 2015). To find out how energy consumption was increased, we measured NAD+/NADH -ratio from the hepatocytes of three human FIGURE 1 | Outline of the Study: Timelines and measurements (A), main phases of study (B), and the acute measurements (C). Frontiers in Aging | www.frontiersin.org October 2021 | Volume 2 | Article 7526364 Hirvonen et al. D-Glyceric Acid Activates Mitochondrial Metabolism donors. The results are scientifically reported for the first time in Supplementary Presentation 3. Statistical Tests and Sufficient Number of Observations Each person was used as their own control. That way individual “noise factors”could be eliminated and we were able to pairwise test whether the intraindividual responses were similar among study persons. Furthermore, to achieve statistically unambiguous results all blinded participants were in the same comparison group during the first week. Pre-assessment on the sufficient group size was based on the results from our pilot tests and related volatilities. Among our relatively homogenous study group with fully comparable intraindividual measurement points, the selected group size turned out to be clearly sufficient. Statistical tests were conducted using IBM SPSS statistics software and Microsoft Excel. Presented statistical test results are mostly from parametric Student’st-tests. When N<15, we checked the normality (Routledge, 2020) of the underlying data using the Kolmogorov-Smirnov test. Non-parametric MannWhitney U-test or SIGN tests were used when clearly needed. p-value <0.05, <0.01, and <0.001 in a one-sided t-test were considered statistically significant, very significant and extremely significant respectively. All presented tests were predetermined or derived from predetermined test settings. FIGURE 2 | Representative energy substrates under the 4-days DGA regimen, 12 h from last DGA dose, and DGA and insulin (A). Schematic flows of all reported plasma energy substrates at whole-body level (B). Statistical tests are based on intra-individual changes (paired t-tests). Error bars are standard errors of the mean (SEM) of the individual changes. Frontiers in Aging | www.frontiersin.org October 2021 | Volume 2 | Article 7526365 Hirvonen et al. D-Glyceric Acid Activates Mitochondrial Metabolism RESULTS AND DISCUSSION The 4and 21-days changes of carefully selected plasma metrics and mRNA expressions from WBCs were used as indirect primary markers for the activation of mitochondrial metabolism. Already after 4 days of the DGA regimen, there is a significant downregulation of all plasma energy substrates (Figure 2). Especially the strong decline in plasma lactate indicates an upregulation of the whole-body mitochondrial (energy) metabolism (Figure 3). In section @ADME and the Timing of the Response of the DGA Activation@, we shortly outline the ADME of DGA and how the response to the DGA regimen materializes from the acute single-dose towards the 4 days administration. The 21-days results show that the whole-body homeostasis has been restored but at a more “energetic”level, the liver and muscle functions having especially improved (Figure 4). Hepatic enhancement is visible from AST and ALT results and muscular improvements from CK results (Figure 5). Low-grade inflammatory markers IL-6 and GlycA were also our ex-antedefined primary markers. Positive results on the reduction of chronic inflammation are presented in Figure 5 and related analyses with hsCRP as an additional proof of concept. Notably, CK, AST, GlycA, and hsCRP showed a strong positive response already to the 4-days DGA regimen (Figures 5A,B,D,E). Day 0 starting values and the 4 days %-changes of all used plasma metrics are ranked by individual VO 2 max results and presented in Supplementary Presentation 1. Cellular level studies on WBCs and hepatocytes are reported in Supplementary Presentation 2, 3 respectively. Acute 45-min measurements on IL-6 and insulin are presented at the end of Supplementary Presentation 1 (“VO 2 max correlating markers2”) and in Supplementary Presentation 4. References are made to supplements when needed to support conclusions. Plasma Energy Substrate Responses to the 4-Days DGA Regimen Plasma energy substrate concentrations reflect intracellular (cytosolic) concentrations via plasma membrane transporters (Figure 3). Main energy substrates like glucose, TGs, and FAs can be restructured and stored in cells and thus are not excreted back into the plasma. Smaller cytosolic metabolites like lactate and pyruvate can be efficiently imported or exported via plasma membrane monocarboxylic acid transporters (Figure 3). FIGURE 3 | Lactate, pyruvate, glucose, and TGs/FAs inflow from plasma and their intracellular metabolism in a cell that can use both FAs and glucose as the main source of energy. Independently of their final use in metabolism (liver or peripheral tissues), the intracellular direction of lactate via pyruvate is towards mitochondria. This is because the cytosolic PK reaction is irreversible. TCA produces most of the NADH for OXPHOS. Glycolysis produces 2 NADH per one glucose. There existed a very strong correlation between the changes of plasma pyruvate and lactate independently of placebo or DGA treatment (>0.90). This correlation strongly indicates that MCTs are able to balance plasma membrane concentration differences efficiently. Frontiers in Aging | www.frontiersin.org October 2021 | Volume 2 | Article 7526366 Hirvonen et al. D-Glyceric Acid Activates Mitochondrial Metabolism Presented substrates in Figure 2A represent all main nutritional categories i.e., fats, carbohydrates, amino acids, ketone bodies, and carboxylic acids. They cover some 90% of used energy substrates at rest. All plasma concentrations of these energy substrates were downregulated by the 4 days DGA regimen (Figure 2A). Observed consistent response extremely likely reflected a decrease in intracellular concentrations of the substrates because the other possibility of increased excretion out of the body from plasma due to the DGA regimen can be sufficiently ruled out by careful organ and substrate-level assessments (Figures 2A,B). Intracellular energy substrate concentrations may decrease through metabolism into either the TCA (Martínez-Reyes and Chandel, 2020), into anabolic reactions, or into intracellular energy stores. All these three metabolic alternatives relate somehow to mitochondrial metabolism because most of the anabolic reactions like glucoand glyceroneogenesis materialize via mitochondria (Figure 3). Also, FA oxidation materializes in mitochondria. Energy Substrate Net Inflow Into Cells Point to Mitochondrial Activation and Increased NADH/NAD + -Ratio We found that the 4-days DGA regimen caused an extremely significant 21% average decline in plasma lactate (p0.0009, Figure 2A). There does not seem to be any deviating persons in lactate responses despite the wide starting range of lactate concentrations (Supplementary Presentation 1, Figure A). When lactate is imported to the cells, its intracellular metabolic routeisalwaysviaLDHandpyruvatetowardsmitochondria (Figure 3). In fact, all presented energy substrates eventually end in mitochondrial metabolism (when oxygen is present). Thus, the results presented in Figure 2A were a strong indication of increased mitochondrial metabolism at the whole-body level. Furthermore, the final catabolism of all energy substrates into CO 2 produces energy-containing NADH molecules. Most of them are produced bytheTCAandusedbytheOXPHOS(Figure 3). All in all, our results show an increase in overall NADH (energy) level in cells via DGA activation. We performed an additional in vitro experiment with cultured hepatocytes. Indeed, NADH generation was significantly activated 3 h after the last DGA dose (Supplementary Presentation 3). The 4-days DGA Regimen Seems to Cause Temporary Hepatic Lactate Shortage The first four bars in Figure 2A represent energy substrates that are mostly provided by the maintaining organs (arrows in Figure 2B) and the last 5 bars (excl. the DGA and insulin) represent substrates that are mostly imported to the liver for glucoand glyceroneogenesis (arrows in Figure 2B). There is an interesting difference between the first 4 and next 5 bars in Figure 2B. Although all are down, the substrates going towards the liver are all statistically significantly or even extremely significantly downregulated, but none of the reductions in the substrates going towards peripheral tissues is significant. It seems that during the 4 days DGA activation a scarcity of glucoand glyceroneogenic plasma substrates towards the liver had clearly developed. Furthermore, from the volume decreases (right-hand scale, horizontal bars in Figure 2A)wecan clearly observe that the scarcity was almost fully due to a decline in lactate. A statistically very significant 21% decline in plasma glycerol (Figure 2A) demonstrates lactate shortage for hepatic glyceroneogenesis. Glycerol is not a direct energy substrate and thus the DGA regimen should not have any major effect on it. Additionally, glycerol kinase enzymes are active mostly in the liver. A robust 21% decline in glycerol indicates that the liver started to import free glycerol from plasma to form glycerol phosphate by hepatic glycerol kinase reactions and to thusly compensate the sharp reduction of plasma lactate. Lactate is the main substrate for hepatic FIGURE 4 | 21 days (A) and 14 days (B) %-changes in selected plasma energy metabolites and insulin. Statistical tests are based on intra-individual changes. In Figure 4A the changes are from Day 0 and in Figure 4B from Day 4. Horizontal bars in bHB and Insulin indicate statistically significant deviation between the placebo and DGA subgroups. Frontiers in Aging | www.frontiersin.org October 2021 | Volume 2 | Article 7526367 Hirvonen et al. D-Glyceric Acid Activates Mitochondrial Metabolism glyceroneogenesis and glycerol phosphate is its product (Nye et al., 2008). Further proof comes from the surprising but temporary decline in plasma glucose, Figure 2A which also points to a mild shortage of lactate. This time for hepatic gluconeogenesis at Day 4 (Figure 2A). Insulin Hinders Glucose Intake to Tissues Likely Because of Increased Intracellular Glucose Storage due to the DGA Activation At the whole-body level, the temporary and benign lactate shortage has likely developed due to increased glycogen (glucose) and TGs/FAs storage into peripheral tissues like skeletal muscles. Plasma glucose level declines statistically almost significantly despite less stimulation from insulin (Figure 2A). One credible explanation in resting measurements was that on top of the lactate shortage also glycogen storage in peripheral tissues was likely to have been increased, as also happens after physical exercise by skeletal muscles (Hermansen et al., 1970;Goodwin, 2010). Also, TGs and FFAs seemed to be used/stored at a slightly higher rate under the DGA regimen (Figures 2A,4A)(Hargreaves and Spriet, 2020). More detailed analysis on plasma TGs and FFAs response to the 4-days DGA regimen is presented in Supplementary Presentation 1 (Figures K,M, and K and M clustered). The 4-Days DGA Regimen Causes Strong Modulation of Mitochondria-Related mRNA Expressions in the Immune Cells The mRNA expressions of certain ATP synthase genes were strongly downregulated at Day4 (Supplementary Presentation 2,Supplementary Presentation 1, Figures B,C). Cellular ATP production is tightly regulated to avoid excessive ATP production. Also, the mRNA expression of the PC gene (Figure 3)was upregulated statistically very significantly during the 4 days DGA regimen (Supplementary Presentation 2, Figure B). In mitochondria the PC enzyme directs incoming energy substrate FIGURE 5 | Enzyme release of creatine kinase (A), aspartate aminotransferase (B), and alanine aminotransferase (C) into the plasma, and low-grade inflammation markers GlycA (D), hsCRP (E), and IL-6 (F). All statistical tests are based on intra-individual changes. SEM error bars are calculated from absolute values. Notes: 1) Day 21 bars in (A–F) are indexed to Day 0 bars so that the Day 21 results fully reflect the changes from the relevant DGA and placebo subgroups at Day 0. 2) There was one clear outlier in the 4-days result in CK and AST from the same participant (see Supplementary Presentation 1, Figures H,I), and one outlier in IL-6 Day 0 results (Supplementary Presentation 1, Figure P). Additionally, in GlycA (Figure 3D) the highest 1/3 ranked by VO 2 max deviated clearly from the other participants and from hsCRP (see Supplementary Presentation 1, Figures N,O). These observations have been excluded to achieve normality of the data. Importantly, even including these outliers, the respective changes in CK, AST, GlycA, and IL-6 would have been statistically significant. 3) The hsCRP data is non-normal due to very high volatility. Due to the high volatility median values of hsCRP are presented in (E) and a non-parametric Sign test was used for the 4-days change. 4) All other bars represent arithmetic means and the intraindividual changes for all others were normally disrtibuted. Statistically more powerful paired t-test was used when comparing the intraindividual changes from Day 0 in (A,B,D, and F). 5) Naturally, when comparing the changes from Day 0 to Day 21 in the placebo group vs. the DGA group (ALT and IL-6, (C,F)), a paired t-test could not be used. Instead, a non-paired t-test was used to compare the changes in the placebo vs. DGA for ALT and IL-6. Frontiers in Aging | www.frontiersin.org October 2021 | Volume 2 | Article 7526368 Hirvonen et al. D-Glyceric Acid Activates Mitochondrial Metabolism