Communication Effects of Short-Term Dietary Protein Restriction on Blood Amino Acid Levels in Young Men
Kim A. Sjøberg 1, Dieter Schmoll 2 , Matthew D. W. Piper 3 , Bente Kiens 1 and Adam J. Rose 4,*
1 Section of Molecular Physiology, Department of Nutrition, Exercise and Sports, Faculty of Science, University of Copenhagen, 2100 Copenhagen, Denmark; [email protected] (K.A.S.); [email protected] (B.K.) 2 Sanofi-Aventis Deutschland GmbH, Industriepark Hoechst, 65926 Frankfurt am Main, Germany; Dieter.Schmoll@sanofi.com 3 School of Biological Sciences, Monash University, Clayton, VIC 3800, Australia; [email protected] 4 Nutrient Metabolism & Signalling Laboratory, Department of Biochemistry and Molecular Biology, Metabolism, Diabetes and Obesity Program, Biomedicine Discovery Institute, Monash University, Clayton 3800, Australia * Correspondence: [email protected]
Received: 7 July 2020; Accepted: 22 July 2020; Published: 23 July 2020
Abstract: Pre-clinical studies show that dietary protein restriction (DPR) improves healthspan and retards many age-related diseases such as type 2 diabetes. While mouse studies have shown that restriction of certain essential amino acids is required for this response, less is known about which amino acids are affected by DPR in humans. Here, using a within-subjects diet design, we examined the effects of dietary protein restriction in the fasted state, as well as acutely after meal feeding, on blood plasma amino acid levels. While very few amino acids were affected by DPR in the fasted state, several proteinogenic AAs such as isoleucine, leucine, lysine, phenylalanine, threonine, tyrosine, and valine were lower in the meal-fed state with DPR. In addition, the non-proteinogenic AAs such as 1- and 3-methyl-histidine were also lower with meal feeding during DPR. Lastly, using in silico predictions of the most limiting essential AAs compared with human exome AA usage, we demonstrate that leucine, methionine, and threonine are potentially the most limiting essential AAs with DPR. In summary, acute meal feeding allows more accurate determination of which AAs are affected by dietary interventions, with most essential AAs lowered by DPR.
Keywords: amino acids; dietary protein; meal feeding; fasting; restriction
1. Introduction Of the dietary macronutrients, protein is pivotal, as the digestion of protein yields amino acids which are vital for life [1,2]. While a severe restriction or a lack of protein is not compatible with life, dietary protein restriction to a certain threshold seems to be beneficial [3–5]. Indeed, dietary protein intake rates are positively related to type 2 diabetes risk [6] as well as all-cause mortality [7] in humans. Furthermore, several pre-clinical studies have demonstrated that dietary protein restriction (DPR) retards several age-related diseases such as type 2 diabetes, Alzheimer’s disease, and perhaps even certain cancers [8–14]. Importantly, several human trials have shown that the basic responses to DPR are conserved from mice to humans [10,13,15]. We and others have shown that the dietary restriction of amino acids can mimic the effects of dietary protein restriction [13,16,17]. Indeed, we recently demonstrated that the restriction of certain amino acids such as threonine and tryptophan was sufficient and necessary to confer the effects of
Nutrients 2020, 12, 2195; doi:10.3390/nu12082195 www.mdpi.com/journal/nutrients Nutrients 2020, 12, 2195 2 of 9
DPR using casein-based diet feeding studies of mice [17]. However, far less is known about which AAs are necessary for the effects of DPR in humans. Thus, to learn more about the effects of DPR in humans, in a pilot study using a within-subjects diet design, we examined the effects of dietary protein restriction on fasting- and meal feeding-induced changes in blood plasma amino acid levels.
2. Materials and Methods
2.1. Human Study Design For this pilot study, five healthy, lean male volunteers, aged 25.6 0.4 years, with a body weight ± of 75.9 5.3 kg (mean SEM), participated. Subjects consumed a controlled diet low in protein ± ± (~9%EP; ~12.8 MJ/d) for 7 days followed by a wash-out period for 7 days on their habitual, mixed diet (~20%EP; ~9.4 MJ/d) and protein intake averaged 0.94 0.03 and 1.57 0.22 g/kg/d in the PR and ± ± habitual diet, respectively (Table1). Blood samples were obtained from the antecubital vein at selected time points in the morning after an overnight fast. A meal test was conducted before as well as at d7 of the experiment, whereby subjects ate an eucaloric meal (i.e., 60 kJ/kg BM containing 15%E or 9%E protein for normal-protein diet (NPD) vs. low-protein diet (LPD), respectively) with venous blood samples taken before as well as at selected times after the meal consumption. All subjects gave informed consent prior to their participation. This study was approved by the Copenhagen Ethics Committee (H-3-2012-129) and was executed in accordance with the code of ethics of the World Medical Association (Helsinki II declaration). Some results of this study have been published previously [10].
Table 1. Diet compositions.
Habitual Diet Protein Restricted Diet Energy, MJ/d 9.4 0.8 12.8 0.6 ± ± Alcohol 0 0 0 0 ± ± Protein, %E 20.2 0.5 9.0 0 ± ± CHO, %E 43.6 0.6 71.0 0 ± ± Fat, %E 36.1 0.9 20.0 0 ± ± Protein, g/d 111 11 73.3 3 ± ± Protein, g/kg BW/d 1.57 0.22 0.97 0.03 ± ± E: caloric energy; CHO: carbohydrate; BW: body weight.
2.2. Blood Plasma Amino Acid Profiling Blood plasma amino acid profiling was conducted using LC–MS/MS. Analysis, including sample preparation and derivatization, was performed using the EZ:faast™ kit (Phenomenex, Torrance, CA, USA) according to the user’s manual. For LC–MS/MS detection, an electrospray ionization-triple quadrupole mass spectrometer (Quantum Ultra, Thermo, Waltham, MA, USA) coupled to a liquid chromatography system (UltiMate3000, Dionex, Sunnyvale, CA, USA) controlled by Xcalibur 2.0.7 software with Dionex Chrom MS link 6.80 was used. Chromatographic separation was achieved on a 250 2 mm EZ:faast AAA–MS column (Phenomenex) at 35 C with a flow rate of 250 µL/min; × ◦ the autosampler temperature was set to 10 ◦C. A sample volume of 1 µL was injected onto the column. Eluents consisted of 10 mM ammonium formate in water (A) and 10 mM ammonium formate in methanol (B). Initial conditions (0 min) were 68% B, then a linear gradient was applied within 13 min to 83% B. The system returned to initial conditions within 4 min and equilibrated for 7 min, resulting in a total run time of 23 min per sample. The column flow was directly converted into the H-electrospray ionization (HESI) source of the mass spectrometer, which was operated in the positive ion mode. Capillary and vaporizer temperatures were maintained at 350 and 50 ◦C, respectively. Sheath gas and auxiliary gas were operated at 40 and 25 (pressure, arbitrary units), and no ion sweep gas was applied. Collision energy and tube lens offset were adjusted accordingly to obtain the highest response for the specific amino acid. Quantification was performed via peak area ratios applied to internal standards provided in the kit. Nutrients 2020, 12, 2195 3 of 9
2.3. In Silico Analyses Nutrients 2020, 12, x FOR PEER REVIEW 3 of 9 Dietary amino acid supply from the low-protein diet was calculated according to Dankost 3000 (Dankost2.3. In ApS, Silico Copenhagen,Analyses Denmark) and compared against the amino acid distribution in the human exome,Dietary as amino previously acid supply described from the [18 low-protein]. Exome matching diet was calculated used the according human proteome to Dankost sequences 3000 downloaded(Dankost fromApS, Copenhagen, Ensembl in 2018Denmark) (GRCh38 and compared assembly). against Briefly, the amino to find acid the distribution average amino in the acid compositionhuman exome, of the exome,as previously we find described the relative [18]. proportionExome matching of each used amino the human acid for proteome each protein. sequences We then computedownloaded the average from relativeEnsembl proportionin 2018 (GRCh38 of each assemb aminoly). acidBriefly, across to find all the proteins average in amino the proteome, acid composition of the exome, we find the relative proportion of each amino acid for each protein. We thus generating an amino acid ratio representative of every gene in the exome. then compute the average relative proportion of each amino acid across all proteins in the proteome, 2.4. Statisticalthus generating Analysis an amino acid ratio representative of every gene in the exome. Statistical2.4. Statistical analyses Analysis were performed using one- or two-way analysis of variance (ANOVA) with t repeated measures,Statistical analyses with Holm–Sidak-adjusted were performed using post-tests. one- or two-way All analyses analysis were of variance carried (ANOVA) out with SigmaPlot with 14 (Systatt repeated Software, measures, Inc., Santawith Holm–Sidak-adjusted Clara, CA, USA) and post-tests. data were All visualized analyses were with GraphPadcarried out Prismwith 8.0 (GraphPadSigmaPlot Software, 14 (Systat Inc., Software, San Diego, Inc.) and CA, data USA). were Statistical visualized details with GraphPad can be found Prism 8.0 within (GraphPad the Figure legends.Software, Differences Inc.). Statistical between details groups can were be found considered within the significant Figure legends. when Differencesp < 0.05. between groups were considered significant when p < 0.05. 3. Results 3. Results 3.1. Effects Of Dietary Protein Restriction on Fasting Venous Blood Plasma Amino Acid Levels 3.1. Effects Of Dietary Protein Restriction on Fasting Venous Blood Plasma Amino Acid Levels A schematic of the study design is shown (Figure1A). Initially, we examined the e ffects of A schematic of the study design is shown (Figure 1A). Initially, we examined the effects of dietary protein restriction on blood amino acid concentrations during fasting. Non-essential (NEAAs; dietary protein restriction on blood amino acid concentrations during fasting. Non-essential (NEAAs; Figure1B–K) and essential (EAAs; Figure1L–T) amino acids are shown. Of the NEAAs, only TYR Figure 1B–K) and essential (EAAs; Figure 1L–T) amino acids are shown. Of the NEAAs, only TYR (Figure(Figure1K) was 1K) significantlywas significantly lower lower in in the the fasted fasted state state withwith LPD. Of Of the the EAAs, EAAs, only only LEU LEU (Figure (Figure 1N) 1N) and PHEand PHE (Figure (Figure1Q) 1Q) were were altered altered with with fasted fasted levels levels lowerlower 7 7 d d after after LPD LPD feeding feeding in the in thefasted fasted state, state, whichwhich were were then then no dinoff erentdifferent to initialto initial levels levels 7 7 d d after after aa NPD feeding. feeding.
Figure 1. Effects of dietary protein restriction on blood serum proteinogenic amino acids during fasting. After an initial sample, young men (n = 5) underwent a 7 d period of consuming a protein-restricted Nutrients 2020, 12, x FOR PEER REVIEW 4 of 9
NutrientsFigure2020, 121. ,Effects 2195 of dietary protein restriction on blood serum proteinogenic amino acids during4 of 9 fasting. After an initial sample, young men (n = 5) underwent a 7 d period of consuming a protein- restricted diet, followed by a 7 d diet matching habitual protein intake (NPD), with blood samples taken in the overnight fasted state before, 3 d and 7 d after LPD, and at 7 d after NPD feeding. (A), diet,schematic followed of the by astudy 7 d diet design. matching The blood habitual drops protein represent intake the (NPD), fasting with samples blood samplesand M represents taken in the an overnightisocaloric fastedmeal tolerance state before, test. 3Blood d and serum 7 d after amino LPD, acid and levels at 7 dincluding after NPD the feeding. non-essential (A), schematic amino acids of the(NEAAs) study design.alanine The(B), arginine blood drops (C), representasparagine the (D fasting), aspartate samples (E), glutamine and M represents (F), glutamate an isocaloric (G), glycine meal tolerance(H), proline test. (I Blood), serine serum (J), and amino tyrosine acid levels (K); and including essentia thel amino non-essential acids (EAAs) amino histidine acids (NEAAs) (L), isoleucine alanine (B(M),), arginine leucine (C(N),), asparagine lysine (O), ( Dmethionine), aspartate (P (E),), phenylalanine glutamine (F), ( glutamateQ), threonine (G), ( glycineR), tryptophan (H), proline (S) and (I), serinevaline ((JT),) andwere tyrosine measured. (K); Data and are essential the mean amino ± SEM. acids One-way (EAAs) RM histidine ANOVA, (L), isoleucinedifferent than (M), time leucine −1: * (Np <), 0.05. lysine (O), methionine (P), phenylalanine (Q), threonine (R), tryptophan (S) and valine (T) were measured. Data are the mean SEM. One-way RM ANOVA, different than time 1: * p < 0.05. ± − InIn additionaddition toto thethe proteinogenicproteinogenic AAs,AAs, non-proteinogenicnon-proteinogenic AAsAAs werewere alsoalso measured.measured. OfOf these,these, nonenone werewere aaffectedffected by by the the diet diet feeding feeding in in the the fasted fasted state state (Figure (Figure2 ).2).
Figure 2. Effects of dietary protein restriction on blood serum non-proteinogenic amino acids Figure 2. Effects of dietary protein restriction on blood serum non-proteinogenic amino acids during during fasting. After an initial sample, young men (n = 5) underwent a 7 d period of consuming fasting. After an initial sample, young men (n = 5) underwent a 7 d period of consuming a protein- a protein-restricted diet, followed by a 7 d diet matching habitual protein intake (NPD) with bloodrestricted samples diet, takenfollowed in theby overnighta 7 d diet matching fasted state habitual before, protein 3 d and intake 7 dafter (NPD) LPD, with and blood at 7 samples d after NPDtaken feeding. in the overnight Blood serum fasted amino state before, acid levels 3 d and including 7 d after 1-methyl LPD, and histidine at 7 d after (A), NPD 3-methyl feeding. histidine Blood (Bserum), α-aminoadipate amino acid levels (C), includingα-aminobutyrate 1-methyl (histidineD), ß-aminoisobutyrate (A), 3-methyl histidine (E), cystine (B), α-aminoadipate (F), citrulline ( (GC),), cystathionineα-aminobutyrate (H), hydroxyproline(D), ß-aminoisobutyrate (I), ornithine (JE),), pyroglutamate cystine (F), (Kcitrulline) and sarcosine (G), cystathionine (L) were measured. (H), Datahydroxyproline are the mean (I), SEM.ornithine (J), pyroglutamate (K) and sarcosine (L) were measured. Data are the mean ± SEM. ± 3.2. Effects of Dietary Protein Restriction on Venous Blood Plasma Amino Acid Levels with Isocaloric Meal3.2. Effects Feeding of Dietary Protein Restriction on Venous Blood Plasma Amino Acid Levels with Isocaloric Meal FeedingWe also conducted isocaloric meal feeding studies before as well as at the end of the 7 d LPD feedingWe period, also conducted which involved isocaloric the subjectsmeal feeding ingesting studie a mixeds before meal as well of 60 as kJ /atkg the BM end containing of the 7 15%d LPD or 9%feeding protein period, for NPD which vs. involved LPD, respectively, the subjects after ingesting an overnight a mixed fast, meal with of blood 60 kJ/kg samples BM takencontaining before 15% as wellor 9% as protein at selected for NPD intervals vs. LPD, after respectively, feeding. With after regard an overnight to NEAAs fast, (Figure with3), blood only TYRsamples (Figure taken3I) before was aasffected, well as both at selected in the fasted intervals state after as well feeding. as at With 60–240 regard min to after NEAAs meal feeding.(Figure 3), With only regard TYR (Figure to EAAs, 3I) allwas three affected, branched both chainin the aminofasted acidsstate as including well as at ILE 60–240 (Figure min4B), after LEU meal (Figure feeding.4C), With and VAL regard (Figure to EAAs,4I) were affected, with levels being lower after meal feeding. In addition, the aromatic AA PHE (Figure4F)
Nutrients 2020, 12, x FOR PEER REVIEW 5 of 9 Nutrients 2020, 12, x FOR PEER REVIEW 5 of 9 all three branched chain amino acids including ILE (Figure 4B), LEU (Figure 4C), and VAL (Figure Nutrients4I)all werethree2020 branchedaffected,, 12, 2195 withchain levels amino being acids lower including after ILEmeal (Figure feeding. 4B), In LEU addition, (Figure the 4C), aromatic and VAL AA (Figure PHE5 of 9 (Figure4I) were 4F) affected, was also with only levels lower being after lower meal afterfeeding. meal Furthermore, feeding. In addition,there was the a mainaromatic effect AA of PHEdiet wasduring(Figure also onlythe 4F) meal was lower feedingalso after only mealtest lower for feeding. the after EAA Furthermore,meal LYS feeding. (Figure there 4D)Furthermore, and was THR a main (Figurethere eff ectwas 4G). of a diet main during effect the of mealdiet feedingduring test the for meal the feeding EAA LYS test (Figure for the4 EAAD) and LYS THR (Figure (Figure 4D)4 andG). THR (Figure 4G).
Figure 3. Effects of dietary protein restriction on blood serum non-essential amino acids during Figure 3. Effects of dietary protein restriction on blood serum non-essential amino acids during isocaloricFigure 3. mealEffects feeding. of dietary Young protein men ( nrestriction = 5) underwent on blood a 7 d serum period non-essential of consuming amino a protein-restricted acids during isocaloric meal feeding. Young men (n = 5) underwent a 7 d period of consuming a protein-restricted diet,isocaloric with isocaloricmeal feeding. meal Young tolerance men tests (n = conducted 5) underwent before a 7 and d period at the ofend consuming of the 7 d aperiod, protein-restricted with blood diet, with isocaloric meal tolerance tests conducted before and at the end of the 7 d period, with blood samplesdiet, with drawn isocaloric at selected meal tolerance time points tests conductedduring the before test. Bloodand at serumthe end amino of the 7acid d period, levels withincluding blood samples drawn at selected time points during the test. Blood serum amino acid levels including alaninesamples ( Adrawn), asparagine at selected (B), aspartatetime points (C), during arginine th (eD test.), glutamine Blood serum (E), glycine amino ( Facid), proline levels ( Gincluding), serine alanine (A), asparagine (B), aspartate (C), arginine (D), glutamine (E), glycine (F), proline (G), serine (alanineH), and ( tyrosineA), asparagine (I) were (B ),measured. aspartate NPD:(C), arginine normal-protein (D), glutamine diet; LPD: (E), low-proteinglycine (F), prolinediet. Data (G ),are serine the (H), and tyrosine (I) were measured. NPD: normal-protein diet; LPD: low-protein diet. Data are the mean(H), and ± SEM. tyrosine Two-way (I) were RM measured. ANOVA, differentNPD: normal-protein than NPD: * pdi Figure 4. Effects of dietary protein restriction on blood serum essential amino acids during isocaloric Figure 4. Effects of dietary protein restriction on blood serum essential amino acids during isocaloric meal feeding. Young men (n = 5) underwent a 7 d period of consuming a protein-restricted diet, mealFigure feeding. 4. Effects Young of dietary men ( nprotein = 5) underwent restriction a on7 d blood period serum of cons essentialuming aamino protein-restricted acids during diet, isocaloric with with isocaloric meal tolerance tests conducted before and at the end of the 7 d period, with blood isocaloricmeal feeding. meal Young tolerance men tests (n = conducted 5) underwent before a 7 and periodd at the of end cons ofuming the 7 da period,protein-restricted with blood diet, samples with samples drawn at selected time points during the test. Blood serum amino acid levels including drawnisocaloric at selected meal tolerance time points tests during conducted the test. before Blood and serum at the aminoend of acidthe 7 levels d period, including with blood histidine samples (A), histidine (A), isoleucine (B), leucine (C), lysine (D), methionine (E), phenylalanine (F), threonine (G), isoleucinedrawn at selected(B), leucine time ( Cpoints), lysine during (D), the methionine test. Blood (E serum), phenylalanine amino acid ( Flevels), threonine including (G), histidine tryptophan (A), tryptophan (H) and valine (I) were measured. NPD: normal-protein diet; LPD: low-protein diet. Data (isoleucineH) and valine (B), leucine(I) were ( Cmeasured.), lysine (D NPD:), methionine normal-protein (E), phenylalanine diet; LPD: low-protein(F), threonine diet. (G ),Data tryptophan are the are the mean SEM. Two-way RM ANOVA, different than NPD: * p < 0.05. A dotted line over the mean(H) and ± SEM. valine± Two-way (I) were RM measured. ANOVA, NPD: different normal-protein than NPD: * dip In addition to the proteinogenic AAs, we also assessed the non-proteinogenic AA (Figure 5). Of Nutrientsthese,2020 only, 12 1MHIS, 2195 (Figure 5A) and 3MHIS (Figure 5B) were affected, with levels lower during 6the of 9 meal feeding. Figure 5. Effects of dietary protein restriction on blood serum non-proteinogenic amino acids during Figure 5. Effects of dietary protein restriction on blood serum non-proteinogenic amino acids during isocaloric meal feeding. Young men (n = 5) underwent a 7 d period of consuming a protein-restricted isocaloric meal feeding. Young men (n = 5) underwent a 7 d period of consuming a protein-restricted diet, with isocaloric meal tolerance tests conducted before and at the end of the 7 d period, with blood diet, with isocaloric meal tolerance tests conducted before and at the end of the 7 d period, with blood samples drawn at selected time points during the test. Blood serum amino acid levels including 1-methyl samples drawn at selected time points during the test. Blood serum amino acid levels including 1- A B α C α D β histidinemethyl ( histidine), 3-methyl (A histidine), 3-methyl ( ), histidine-aminoadipate (B), α-aminoadipate ( ), -aminobutyrate (C), α-aminobutyrate ( ), -aminoisobutyrate (D), β- (E),aminoisobutyrate cystine (F), citrulline (E), cystine (G), cystathionine (F), citrulline ( H(G),), hydroxyproline cystathionine (H (I),), hydroxyproline ornithine (J), pyroglutamate (I), ornithine ( (JK),) andpyroglutamate sarcosine (L) were(K) and measured. sarcosine NPD: (L) were normal-protein measured. diet;NPD: LPD: normal-p low-proteinrotein diet.diet; DataLPD: arelow-protein the mean SEM. Two-way RM ANOVA, different than NPD: * p < 0.05. ± diet. Data are the mean ± SEM. Two-way RM ANOVA, different than NPD: * p < 0.05. 3.3. Human AA Exome Comparision Indentifies Potentially Limiting Essential Amino Acids With DPR 3.3. Human AA Exome Comparision Indentifies Potentially Limiting Essential Amino Acids With DPR Previously, it was shown that one can predict which AAs will be most limiting in a particular Previously, it was shown that one can predict which AAs will be most limiting in a particular diet by exome AA abundance [18]. Hence, we examined this. For this, we calculated the intake of diet by exome AA abundance [18]. Hence, we examined this. For this, we calculated the intake of each AA from the LPD (Figure6A) as well as the %AA abundance in the human exome (Figure6B). each AA from the LPD (Figure 6A) as well as the %AA abundance in the human exome (Figure 6B). A comparison of this demonstrated that certain AAs such as GLN/GLU, ASN/ASP, ARG and PHE A comparison of this demonstrated that certain AAs such as GLN/GLU, ASN/ASP, ARG and PHE areare abundant abundant in in the the LPD LPD relative relative to to % % representation representationin inthe the humanhuman AAAA exomeexome and thus unlikely to to bebe limiting limiting (Figure (Figure6C). 6C). However, However, some some NEAAs NEAAs such such as as CYS, CYS, GLY, GLY, ALA, ALA, and and SERSER werewere potentiallypotentially limiting.limiting. In In addition, addition, EAAs EAAs such such as as MET, MET, THR, THR, and and LEULEU werewere alsoalso limiting,limiting, with TRP and and LYS LYS being being closeclose to to limiting limiting (Figure (Figure6C). 6C). Nutrients 2020, 12, 2195 7 of 9 Nutrients 2020, 12, x FOR PEER REVIEW 7 of 9 Figure 6. Identification of leucine, methionine, and threonine as potentially limiting essential amino acidsFigure with 6. Identification a low-protein of intake. leucine, A: methionine, calculated and daily th proteinogenicreonine as potentially amino acidlimiting intake essential rates during amino low-proteinacids with a dietlow-protein feeding. intake. B: percent A: calculated representation daily ofproteinogenic AA abundance amino in theacid human intake exome.rates during C: scatter low- plotprotein of AA diet intake feeding. vs. %AAB: percent in the representation human exome. of The AA dashedabundance line dissectsin the human the center exome. of THR, C: scatter the most plot limitingof AA intake EAA. vs. %AA in the human exome. The dashed line dissects the center of THR, the most limiting EAA. 4. Discussion 4. DiscussionMultiple studies have shown that dietary protein restriction promotes metabolic health via restrictionMultiple of dietary studies amino have acids.shown In that particular, dietary weprotein have recentlyrestriction shown promotes that restriction metabolic of health essential via aminorestriction acids of is dietary both su ffiaminocient acids. and necessary In particular, to confer we thehave metabolic recently remodeling shown that that restriction occurs with of essential dietary proteinamino restrictionacids is both (DPR) sufficient in mice and [17], necessary less information to confer about the which metabolic amino remodeling acids do so inthat humans. occurs Here,with wedietary show protein that, particularly restriction during(DPR) mealin mice feeding [17], tests,less information the blood plasma about levels which of amino essential acids amino do acidsso in isoleucine,humans. Here, leucine, we valine show (i.e., that, branched particularly chain during AA), lysine, meal phenylalanine,feeding tests, andthe threonine,blood plasma as well levels as the of non-essentialessential amino AA acids tyrosine, isoleucine, were lowerleucine, in youngvaline (i.e., men branched undergoing chain DPR AA), compared lysine, phenylalanine, with their habitual and diet.threonine, A similar as well signature as the was non-essential found in our AA former tyrosine, studies were of micelower [ 9in,17 young], demonstrating men undergoing mammalian DPR conservationcompared with of thesetheir habitual responses. diet. Additionally, A similar signature this AA was signature found is in reminiscent our former ofstudies several of studiesmice [9,17], that havedemonstrating shown higher mammalian blood levels conservati of theseon amino of these acids asresponses. predictive Additi of currentonally, or futurethis AA type signature 2 diabetes is riskreminiscent in humans of [several19–21]. studies Given thatthat dietaryhave shown protein higher intake blood positively levels correlates of these amino with T2D acids risk as [predictive6,22], it is thenof current possible or that future this type signature 2 diabetes is reflective risk ofin thishumans association. [19–21]. Given that dietary protein intake positivelyWe have correlates preciously with demonstrated T2D risk [6,22], in mice, it is usingthen possible casein-based that diets,this signature that the restrictionis reflective of EAAs,of this particularlyassociation. THR and TRP, was necessary and sufficient to confer the metabolic effects of DPR [17]. Thus,We even have though preciously certain demonstrated NEAAs were in deemed mice, using to be ca thesein-based most limiting diets,according that the restriction to in silico of exomeEAAs, matchingparticularly (Figure THR6 and), this TRP, is likely was necessary to be inconsequential and sufficient [ 17 to]. confer LEU, METthe metabolic and THR effects were shownof DPR to [17]. be theThus, most even limiting though EAAs certain (Figure NEAAs6). were However, deemed of these, to be the only most the bloodlimiting levels according of LEU to and in silico THR exome were amatchingffected by (Figure low-protein 6), this feeding is likely (Figure to be 4inconsequential), and levels of LEU[17]. LEU, and MET MET can and be THR metabolically were shown spared to be inthe vivo mostby limiting amino acidsEAAs which(Figure share 6). However, a similar of metabolic these, only pathway the blood such levels as isoleucine of LEU and and THR cysteine, were respectivelyaffected by low-protein [13,23]. Thus, feeding given (Figure that THR 4), and was levels the most of LEU limiting and EAAMET identifiedcan be metabolically (Figure6) and spared its bloodin vivo levels by amino were lower acids with which DPR share (Figure a similar4), it is likelymetabolic that thepathway restriction such of as THR isoleucine alone to levelsand cysteine, within DPRrespectively would mimic[13,23]. the Thus, effects given of DPR.that THR Indeed, was THR the most restriction limiting without EAA totalidentified AA restriction (Figure 6) mimicsand its theblood eff ectslevels of were DPR lower in mice, with without DPR (Figure the negative 4), it is side-e likelyff ectsthat ofthe reduced restriction skeletal of THR muscle alone mass to levels [17]. Inwithin addition, DPR wewould previously mimic demonstratedthe effects of DPR. that dietary Indeed, THR THR restriction restriction improves without metabolic total AA healthrestriction in a pre-clinicalmimics the mouseeffects of model DPR ofin obesitymice, without driven the type nega 2 diabetestive side-effects [17]. Thus, of reduce futured studies skeletal should muscle closely mass examine[17]. In addition, the effects we dietary previously threonine demonstrated restriction that in humans.dietary THR restriction improves metabolic health in a Itpre-clinical is worth noting mouse that model the e offfects obesity of DPR driven on certain type 2 blood diabetes plasma [17]. AA Thus, levels future were studies only observed should withclosely the examine acute meal the effects feeding dietary test. Thisthreonine result restriction is congruent in humans. with the observation that differences in biomarkersIt is worth in response noting that to dietary the effects interventions of DPR on are ce bestrtain observed blood plasma with acute AA challengeslevels were such only as observed feeding orwith fasting the acute [24,25 meal]. In ourfeeding opinion, test. theseThis result acute nutritionalis congruent interventions with the observation allow for biggerthat differences effect sizes, in andbiomarkers less scatter, in response of biomarker to dietary levels interventions within study groups.are best Thus,observed we concludewith acute that challenges it is advisable such toas conductfeeding or such fasting nutritional [24,25]. ‘synchronization’ In our opinion, these in dietary acute interventions.nutritional interventions allow for bigger effect sizes,There and less are scatter, several of limitations biomarker of levels our work within that study should groups. be noted. Thus, Wewe conclude used a small, that homogenousit is advisable population—youngto conduct such nutritional Danish ‘synchronization’ men. Thus, whether in dietary similar interventions. responses would be found with humans of differentThere age, are sex several and ethnicity limitations warrants of our furtherwork that investigation. should be noted. The study We used design a small, was a homogenous longitudinal population—young Danish men. Thus, whether similar responses would be found with humans of Nutrients 2020, 12, 2195 8 of 9 fixed-order diet design, and thus future studies should consider using a randomized cross-over diet design. With the low-protein feeding, subjects consumed more energy overall, and particularly the consumption of both carbohydrates and fats also differed. Thus, we cannot formally rule out that the effects that we observe are directly related to the level of protein in the diet. Lastly, our subject number was rather low, and we acknowledge the possibility of false-positive/negative results due to the small sample size. 5. Conclusions In summary, compared with measuring in the fasted state, acute meal feeding allows more accurate determination of which AAs are affected by dietary interventions, with most essential AAs lowered by DPR. In addition, those essential AAs predicted to be limiting by exome matching are indeed lower with DPR upon meal feeding, indicating that this method may be used to predict those essential AAs that are limiting in a particular diet. Author Contributions: Conceptualization, A.J.R.; methodology, D.S., M.D.W.P., and B.K.; formal analysis, M.D.W.P., B.K., and A.J.R.; investigation, K.A.S., D.S., and B.K.; resources, D.S. and B.K.; data curation, B.K. and A.J.R.; writing—original draft preparation, A.J.R.; writing—review and editing, K.A.S., D.S., M.D.W.P., B.K., and A.J.R.; visualization, A.J.R.; supervision, B.K.; project administration, B.K. and A.J.R.; funding acquisition, B.K. and A.J.R. All authors have read and agreed to the published version of the manuscript. Funding: AJR: EFSD Lilly project award. BK: Novo Nordisk Foundation (NNF17OC0029054). MP: ARC FT150100237. Acknowledgments: We gratefully acknowledge the time and efforts of the participants of this study. Conflicts of Interest: D.S. is an employee of Sanofi-Aventis Deutschland GmbH, a pharmaceutical company. All other authors declare no conflict of interest. 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