Abstract
When neonatal pigs continuously fed formula are supplemented with leucine pulses, muscle protein synthesis and body weight gain are enhanced. To identify the responsible mechanisms, we combined plasma metabolomic analysis with transcriptome expression of the transcriptome and protein catabolic pathways in skeletal muscle. Piglets (n = 23, 7-day-old) were fed continuously a milk replacement formula via orogastric tube for 21 days with an additional parenteral infusion (800 μmol kg−1 h−1) of either leucine (LEU) or alanine (CON) for 1 h every 4 h. Plasma metabolites were measured by liquid chromatography-mass spectrometry. Gene and protein expression analyses of longissimus dorsi muscle were performed by RNA-seq and Western blot, respectively. Compared with CON, LEU pigs had increased plasma levels of leucine-derived metabolites, including 4-methyl-2-oxopentanoate, beta-hydroxyisovalerate, β-hydroxyisovalerylcarnitine, and 3-methylglutaconate (P ≤ 0.05). Leucine pulses downregulated transcripts enriched in the Kyoto Encyclopedia of Genes and Genomes terms “spliceosome,” “GAP junction,” “endocytosis,” “ECM-receptor interaction,” and “DNA replication”. Significant correlations were identified between metabolites derived from leucine catabolism and muscle genes involved in protein degradation, transcription and translation, and muscle maintenance and development (P ≤ 0.05). Further, leucine pulses decreased protein expression of autophagic markers and serine/threonine kinase 4, involved in muscle atrophy (P ≤ 0.01). In conclusion, results from our studies support the notion that leucine pulses during continuous enteral feeding enhance muscle mass gain in neonatal pigs by increasing protein synthetic activity and downregulating protein catabolic pathways through concerted responses in the transcriptome and metabolome.
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Abbreviations
- AA:
-
Amino acids
- ABLIM3:
-
Actin binding LIM protein family member 3
- ALDH4A1:
-
Aldehyde dehydrogenase 4 family member A1
- AMPKα:
-
AMP-activated protein kinase α
- ANKH:
-
ANKH inorganic pyrophosphate transport regulator
- ANKRD6:
-
Ankyrin repeat domain 6
- AP4S1:
-
Adaptor related protein complex 4 sigma 1 subunit
- ASF1B:
-
Anti-silencing function 1B histone chaperone
- AU:
-
Arbitrary units
- BCAA:
-
Branched-chain amino acids
- BCL-XL:
-
B-cell lymphoma-extra large
- BW:
-
Body weight
- CCNJL:
-
Cyclin J like
- CD9:
-
CD9 molecule
- CDCA2:
-
Cell division cycle associated 2
- CHRNA3:
-
Cholinergic receptor nicotinic alpha 3 subunit
- CON:
-
Control group
- DAVID:
-
Database for Annotation, Visualization, and Integrated Discovery
- DEGs:
-
Differentially expressed genes
- DSCC1:
-
DNA replication and sister chromatid cohesion 1
- EAA:
-
Essential amino acids
- ERLEC1:
-
Endoplasmic reticulum lectin 1
- ESF1:
-
ESF1 nucleolar pre-rRNA processing protein, homolog
- FN1:
-
Fibronectin 1
- FOXO:
-
Forkhead box protein O
- GNRHR2:
-
Gonadotropin-releasing hormone II receptor
- GO:
-
Gene ontology
- GPR37L1:
-
G protein-coupled receptor 37 like 1
- GRM7:
-
Glutamate metabotropic receptor 7
- HAUS1:
-
HAUS augmin like complex subunit 1
- HMGCLL1:
-
3-Hydroxymethyl-3-methylglutaryl-CoA lyase like 1
- INO80:
-
Chromatin-remodeling ATPase INO80
- KEGG:
-
Kyoto encyclopedia of genes and genomes
- KIC:
-
α-Ketoisocaproic acid
- KLHDC3:
-
Kelch domain containing 3
- LC3I/II:
-
Microtubule-associated protein 1A/1B light chain 3A
- LD:
-
Longissimus dorsi
- LEU:
-
Leucine supplemented group
- LPXN:
-
Leupaxin
- MAFbx:
-
Muscle Atrophy F-Box/Atrogin-1
- MFAP3L:
-
Microfibrillar associated protein 3 like
- MMP15:
-
Matrix metallopeptidase 15
- mTORC1:
-
Mammalian target of rapamycin complex 1
- MuRF-1:
-
Muscle RING-finger protein-1
- NUAK2:
-
NUAK family SNF1-like kinase 2
- OFD1:
-
Oral-facial-digital syndrome 1 protein
- OPHN1:
-
Oligophrenin 1
- OPTN:
-
Optineurin
- P/T:
-
Phosphorylated/total protein
- PARP8:
-
Protein mono-ADP-ribosyltransferase
- PC:
-
Principal component analysis
- PDE10A:
-
Phosphodiesterase 10A
- PKB/Akt:
-
Protein kinase B
- POPDC3:
-
Popeye domain containing 3
- REPS2:
-
RalBP1-associated Eps domain-containing 2
- RGS3:
-
Regulator of G-protein signaling 3
- RNA-seq:
-
High-throughput RNA sequencing
- RNF17:
-
Ring finger protein 17
- ROMO1:
-
Reactive oxygen species modulator 1
- RPKM:
-
Reads per kilobase per million mapped reads
- SERBP1:
-
Plasminogen activator inhibitor 1 RNA-binding protein
- SF3B4:
-
Splicing factor 3b subunit 4
- SIRT-1:
-
Sirtuin 1
- SNX32:
-
Sorting nexin 32
- STK4:
-
Serine/threonine kinase 4
- TAS2R40:
-
Taste 2 receptor member 40
- TBX21:
-
T-box 21
- TMEM117:
-
Transmembrane protein 117
- UBL4A:
-
Ubiquitin like 4A
- VNN2:
-
Vanin 2
- ZBTB32:
-
Zinc finger and BTB domain containing 32
- ZNF207:
-
Zinc finger protein 207
- ZNF385D:
-
Zinc finger protein 385D
- ZNF652:
-
Zinc finger protein 652
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Conceptualization—CBR, MF and TD. Data curation—CBR, AS and HN. Formal analysis—RM, BP, AC, MM, and MI. Funding acquisition—MF and TD. Investigation—CBR, AS, HN, MF and TD. Methodology—CBR, AS and TD. Project administration—MF and TD. Resources—MF and TD. Supervision—MF and TD. Validation—MF and TD. Visualization—RM, BP, AC, MM, and MI. Writing—original draft—RM, BP, AC, MM, and MI. Writing—review and editing—all authors.
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Experiments were carried out in accordance with the Institutional Animal Care and Use Committee of Baylor College of Medicine and conducted in accordance with the National Research Council’s Guide for the Care and Use of Laboratory Animals (2011).
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726_2020_2894_MOESM1_ESM.xlsx
Online Resource 1 Metabolomic analysis results in plasma from neonatal pigs fed continuously with an orogastric tube for 21 days and supplemented with either leucine or alanine pulses parenterally at 4-h intervals (XLSX 180 kb)
726_2020_2894_MOESM2_ESM.xlsx
Online Resource 2 Differentially expressed genes (P ≤ 0.05) in skeletal muscle tissue from neonatal pigs fed continuously with an orogastric tube for 21 days and supplemented with either leucine or alanine pulses parenterally at 4-h intervals (XLSX 113 kb)
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Manjarín, R., Boutry-Regard, C., Suryawan, A. et al. Intermittent leucine pulses during continuous feeding alters novel components involved in skeletal muscle growth of neonatal pigs. Amino Acids 52, 1319–1335 (2020). https://doi.org/10.1007/s00726-020-02894-5
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DOI: https://doi.org/10.1007/s00726-020-02894-5