Skeletal muscle amino acid uptake is lower and alanine production is greater in late gestation intrauterine growth-restricted fetal sheep hindlimb

Abstract

In a sheep model of intrauterine growth restriction (IUGR) produced from placental insufficiency, late gestation fetuses had smaller skeletal muscle mass, myofiber area, and slower muscle protein accretion rates compared with normally growing fetuses. We hypothesized that IUGR fetal muscle develops adaptations that divert amino acids (AAs) from protein accretion and activate pathways that conserve substrates for other organs. We placed hindlimb arterial and venous catheters into late gestation IUGR (n = 10) and control (CON, n = 8) fetal sheep and included an external iliac artery flow probe to measure hindlimb AA uptake rates. Arterial and venous plasma samples and biceps femoris muscle were analyzed by mass spectrometry-based metabolomics. IUGR fetuses had greater abundance of metabolites enriched within the alanine, aspartate, and glutamate metabolism pathway compared with CON. Net uptake rates of branched-chain AA (BCAA) were lower by 42%-73%, and muscle ammoniagenic AAs (alanine, glycine, and glutamine) were lower by 107%-158% in IUGR hindlimbs versus CON. AA uptake rates correlated with hindlimb weight; the smallest hindlimbs showed net release of ammoniagenic AAs. Gene expression levels indicated a decrease in BCAA catabolism in IUGR muscle. Plasma purines were lower and plasma uric acid was higher in IUGR versus CON, possibly a reflection of ATP conservation. We conclude that IUGR skeletal muscle has lower BCAA uptake and develops adaptations that divert AAs away from protein accretion into alternative pathways that sustain global energy production and nitrogen disposal in the form of ammoniagenic AAs for metabolism in other organs.

Keywords: fetal growth restriction; metabolomics; skeletal muscle.

Fig. 1.

Metabolomic profiles of skeletal muscle of intrauterine growth restriction (IUGR) and control (CON) fetuses. A: partial least squares discriminant analysis was used to identify metabolites with changes in abundance that defined separation of samples between the IUGR (n = 10 fetuses) and CON (n = 8 fetuses) groups. B: heat map of the 30 metabolites with the highest variable importance in projection scores. Each square is representative of the mean levels of that metabolite for the individual animal. Row values are normalized for each metabolite, and quantitative changes are color coded from red (high) to blue (low). Student’s t test was conducted to detect differences in fold change. *False discovery rate-adjusted P ≤ 0.05.

Fig. 2.

Selected features to highlight the tricarboxylic acid (TCA) cycle; alanine, aspartate, and glutamate metabolism; and other key metabolites. A: metabolites from intrauterine growth restriction (IUGR; n = 10 fetuses) and control (CON; n = 8 fetuses) fetal skeletal muscle. B: features from IUGR (shaded box) and CON (open box) fetal arterial (IUGR-A, CON-A) and venous (IUGR-V, CON-V) plasma samples. Data are from MetaboAnalyst as presented in Figs. 1 and 3 and analyzed using Student’s t test (A) or two-way ANOVA (B). *False discovery rate-adjusted P ≤ 0.1 by Tukey’s post hoc test. Box denotes 25th and 75th percentiles; bars denote minimum and maximum values. A, arterial; V, venous.

Fig. 3.

Metabolomic profiles of arterial and venous plasma of intrauterine growth restriction (IUGR) and control (CON) fetuses. A: partial least squares-discriminant analysis was used to identify metabolites with changes in abundance that defined separation of samples between the IUGR (n = 10 fetuses) and CON (n = 8 fetuses) groups. B: heat map of the 30 metabolites with the highest variable importance in projection scores. For simplicity, each square is representative of the mean levels of that metabolite per group and vessel. Row values are normalized for each metabolite, and quantitative changes are color coded from red (high) to blue (low). A, arterial plasma; FBP, fructose 1,6-bisphosphate; G3P, sn-glycerol 3-phosphate; NAML, N-Acyl-d-mannosaminolactone; THcHDO, 3D-(3,5/4)-trihydroxycyclohexane-1,2-dione; V, venous plasma. ANOVA was performed on all 4 groups (CON-A, CON-V, IUGR-A, and IUGR-V). Student’s t test was performed separately on the arterial and venous plasma of IUGR versus CON. *False discovery rate-adjusted P ≤ 0.05.

Fig. 4.

Reduced hindlimb uptake of essential and nonessential amino acids in the intrauterine growth restriction (IUGR) fetus. A: total amino acid (AA) uptake rates. B: individual AA uptake rates. Data are represented as means ± SE; IUGR (n = 8) and control (CON; n = 8). Arg, arginine; Asp, aspartate; ASPG, asparagine; Cys, cysteine; Glu, glutamate; His; Lys, lysine; Met, methionine; Orn, ornithine; Phe, phenylalanine; Pro, proline; Ser, serine; Tau, taurine; Thr, threonine; Trp, tryptophan; Tyr, tyrosine. *Statistical significance at P ≤ 0.05 vs. CON, Student’s t test. C: linear relationship between hindlimb weight and alanine (Ala), glutamine (Gln), glycine (Gly), leucine (Leu), isoleucine (Ile), and valine (Val) uptake rates. IUGR fetuses and CON fetuses with R² and P values are shown.

Fig. 5.

Gene expressions of enzymes in the fetal skeletal muscle. A: genes involving in the glycolysis and tricarboxylic acid (TCA) cycle pathways. B: branched-chain amino acid catabolism in the intrauterine growth restriction (IUGR; n = 10) versus control (CON; n = 8). *P ≤ 0.05, unpaired Student’s t tests. ALT, alanine aminotransaminase 1; AST, aspartate transaminase; BCAT1, branched-chain amino acid transaminase 1; BCAT2, branched-chain amino acid transaminase 2; BCKD, branched-chain α-keto acid dehydrogenase; BCKDK, branched-chain ketoacid dehydrogenase kinase; GS, glutamine synthetase; GLS, glutaminase; LDHA, lactate dehydrogenase A; LDHB, lactate dehydrogenase B; PC, pyruvate carboxylase; PDH, pyruvate dehydrogenase; PDK4, pyruvate dehydrogenase kinase 4; PFK, phosphofructokinase; PK, pyruvate kinase.

Fig. 6.

Pathway diagram of glycolysis, pentose phosphate pathway (PPP), and tricarboxylic acid (TCA) cycle highlighting metabolites that differed between control (CON) and intrauterine growth restriction (IUGR) fetal skeletal muscle and plasma. Selected metabolites are shown from the top 30 variable importance in projection scores that were higher (green), lower (red), or unchanged (black) in IUGR (n = 10) fetal skeletal muscle (M) and/or femoral arterial and venous plasma (AV) when compared with CON (n = 8) fetuses. The gene expression of selected enzymes (italics) in the fetal skeletal muscle also are shown. PPP production of ribose was lower in IUGR muscle and AV, as was adenosine in IUGR muscle for DNA/RNA and ATP production. Despite lower glycolytic intermediates in IUGR AV, pyruvate concentrations were higher in IUGR muscle. LDHA, LDHB, and PDH expressions was lower in IUGR muscle, and alanine concentrations were higher in IUGR AV, supporting alanine release from muscle, as opposed to lactate production or conversion of pyruvate to acetyl-CoA for entry into the TCA cycle. Lysine and its catabolic product α-hydroxyglutarate were higher in IUGR muscle, yet α-ketoglutarate concentrations were unchanged; leucine/isoleucine concentrations also were higher in IUGR muscle, yet BCAT expression was lower, further supporting limited substrate entry into the TCA cycle in IUGR muscle. Finally, intermediates in the arginine synthesis pathway were lower in IUGR muscle. ALT, alanine aminotransaminase 1; BCAT1, branched-chain amino acid transaminase 1; BCAT2, branched-chain amino acid transaminase 2; FBP, fructose 1,6-bisphosphate; G3P, sn-glycerol 3-phosphate; LDHA, lactate dehydrogenase A; LDHB, lactate dehydrogenase B; PC, pyruvate carboxylase; PDH, pyruvate dehydrogenase; PDK4, pyruvate dehydrogenase kinase 4; PFK, phosphofructokinase; PK, pyruvate kinase.

Fig. 7.

Summary of proposed metabolic adaptations in intrauterine growth restriction (IUGR) skeletal muscle that conserve energy utilization and result in the release of ammoniagenic amino acids (AAs; alanine, glutamine, glycine) and uric acid. Metabolites, AA uptake rates, and gene expressions of enzymes (italics) in the IUGR (n = 10) fetal skeletal muscle or plasma that were higher (green), lower (red), or unchanged (black) when compared with control (CON; n = 8) fetuses. Black and gray-shaded arrows and font show proposed increases and decreases, respectively, in metabolic pathway flux. Based on these analyses, we propose that reduced oxidative metabolism (through glycolysis and the TCA cycle) and reduced availability of phosphate and adenosine result in increased entry of ADP into a salvage pathway for ATP generation. AMP and ammonia are then overly produced in IUGR muscle. AMP is further processed to uric acid via AMP deaminase, and excess ammonia is converted and released into the plasma in the forms of glutamine and glycine. Alanine, which is a gluconeogenic substrate and released into the circulation, is produced as a result of a series of transamination reactions that alleviate the load of pyruvate and glutamate. α-KG, α-ketoglutarate; ADA, adenosine deaminase; BCAA, branched-chain amino acid; BCKA, branched-chain α-ketoacids; GCS, γ-glutamylcysteine synthetase; GLS, glutaminase; GS, glutamine synthetase; Pi, orthophosphate; PPi, diphosphate.