Concordance of changes in metabolic pathways based on plasma metabolomics and skeletal muscle transcriptomics in type 1 diabetes

Tumpa Dutta, High Seng Chai, Lawrence E Ward, Aditya Ghosh, Xuan-Mai T Persson, G Charles Ford, Yogish C Kudva, Zhifu Sun, Yan W Asmann, Jean-Pierre A Kocher, K Sreekumaran Nair, Tumpa Dutta, High Seng Chai, Lawrence E Ward, Aditya Ghosh, Xuan-Mai T Persson, G Charles Ford, Yogish C Kudva, Zhifu Sun, Yan W Asmann, Jean-Pierre A Kocher, K Sreekumaran Nair

Abstract

Insulin regulates many cellular processes, but the full impact of insulin deficiency on cellular functions remains to be defined. Applying a mass spectrometry-based nontargeted metabolomics approach, we report here alterations of 330 plasma metabolites representing 33 metabolic pathways during an 8-h insulin deprivation in type 1 diabetic individuals. These pathways included those known to be affected by insulin such as glucose, amino acid and lipid metabolism, Krebs cycle, and immune responses and those hitherto unknown to be altered including prostaglandin, arachidonic acid, leukotrienes, neurotransmitters, nucleotides, and anti-inflammatory responses. A significant concordance of metabolome and skeletal muscle transcriptome-based pathways supports an assumption that plasma metabolites are chemical fingerprints of cellular events. Although insulin treatment normalized plasma glucose and many other metabolites, there were 71 metabolites and 24 pathways that differed between nondiabetes and insulin-treated type 1 diabetes. Confirmation of many known pathways altered by insulin using a single blood test offers confidence in the current approach. Future research needs to be focused on newly discovered pathways affected by insulin deficiency and systemic insulin treatment to determine whether they contribute to the high morbidity and mortality in T1D despite insulin treatment.

Figures

FIG. 1.
FIG. 1.
Heat map analysis of plasma metabolites in T1D during insulin deficiency (I−) and insulin treatment (I+) and comparison with ND. Metabolite perturbations in plasma were calculated based on the median for each metabolite level of three independent biological replicates of plasma samples from each study participant. Each row represents a metabolite, and each column depicts a subject. The study groups are color coded as follows: insulin-deprived (I−) T1D is denoted in blue, insulin-treated (I+) T1D is denoted in red, and ND groups are denoted in maroon. The fold change in metabolite levels is color coded: red pixels, upregulation; blue, downregulation; yellow, no significant change. Metabolites such as acetate, lactate, acetoacetate, hydroxybutyrate, gluconate, hydroxy adipate, carnitines, glucosamine, and taurocholate including amino acid (e.g., leucine, isoleucine, valine, N-methyl histidine, keto glutarate, glutamate, alanine, phenylalanine) were all found to be elevated in I− T1D (Supplementary Table 4). A consistent decrease was observed in other metabolites, e.g., hydroxypyridine, nicotinamide, hydroxyl nicotinic acid, adipate, methylthioribose, uridine, xanthine, hypoxanthine, methylguanosine, N-acetyl tryptophan, pipecolate, homoserine, aldosterone, arachidonyl lysolecithin, phosphoethanolamine, etc.
FIG. 2.
FIG. 2.
A: The effect of differential regulation of metabolites on the canonical pathways during I− in T1D in comparison with I+ T1D and ND. The significance of the pathways was evaluated using P values and false discovery rate <0.05. B: Altered canonical pathways following insulin treatment in T1D in comparison with ND. *Metabolic pathways that were observed exclusively after systemic insulin treatment. The significance of the pathways was evaluated using P values and false discovery rate <0.05. nNOS, neuronal nitric oxide synthase; PDGF, platelet-derived growth factor; TCA, tricarboxylic acid; GPCR, G protein-coupled receptor; FM, function and metabolism.
FIG. 2.
FIG. 2.
A: The effect of differential regulation of metabolites on the canonical pathways during I− in T1D in comparison with I+ T1D and ND. The significance of the pathways was evaluated using P values and false discovery rate <0.05. B: Altered canonical pathways following insulin treatment in T1D in comparison with ND. *Metabolic pathways that were observed exclusively after systemic insulin treatment. The significance of the pathways was evaluated using P values and false discovery rate <0.05. nNOS, neuronal nitric oxide synthase; PDGF, platelet-derived growth factor; TCA, tricarboxylic acid; GPCR, G protein-coupled receptor; FM, function and metabolism.
FIG. 3.
FIG. 3.
Comparison of plasma metabolome with transcriptome of I− versus ND (A) and I+ versus ND (B). The coclustering between the metabolomic changes and the transcripts of the corresponding muscle genes showed similar directional changes on the canonical pathways, although the statistical significance was different. The microarray/transcriptome data and the metabolome data are marked with an orange bar and blue bar, respectively. *Pathways used to build metabolic networks, as shown in Fig 4. EGF, epidermal growth factor; ERK, extracellular signal–related kinase; nNOS, neuronal nitric oxide synthase; PDGF, platelet-derived growth factor; TGF, transforming growth factor; GPCR, G protein-coupled receptor; LTD4, leukotriene receptor D4; HGF, hepatocyte growth factor; EMT, epithelial mesenchymal transition; VDR, vitamin D3 receptor; fMLP, N-formylated peptides like fMLP.
FIG. 3.
FIG. 3.
Comparison of plasma metabolome with transcriptome of I− versus ND (A) and I+ versus ND (B). The coclustering between the metabolomic changes and the transcripts of the corresponding muscle genes showed similar directional changes on the canonical pathways, although the statistical significance was different. The microarray/transcriptome data and the metabolome data are marked with an orange bar and blue bar, respectively. *Pathways used to build metabolic networks, as shown in Fig 4. EGF, epidermal growth factor; ERK, extracellular signal–related kinase; nNOS, neuronal nitric oxide synthase; PDGF, platelet-derived growth factor; TGF, transforming growth factor; GPCR, G protein-coupled receptor; LTD4, leukotriene receptor D4; HGF, hepatocyte growth factor; EMT, epithelial mesenchymal transition; VDR, vitamin D3 receptor; fMLP, N-formylated peptides like fMLP.
FIG. 4.
FIG. 4.
Integration of the metabolomics with transcriptomics data and their superimposition to build metabolic networks. A: Metabolic network of PPAR transcription pathway, which is connected to other metabolic processes such as lipid homeostasis, glucose, fatty acid metabolism, and inflammatory response. B: Network model of downstream of insulin-signaling pathways. The metabolites and the gene names shown in red are upregulated, and the same shown in blue are downregulated during insulin deficiency. B, binding; C, cleavage; CoA, coenzyme A; Erk, extracellular signal–related kinase; HPODE, hydroperoxylinoleic acid; IE, influence on expression; MAP, mitogen-activated protein; MAPK, MAP kinase; PDGF, platelet-derived growth factor; PI3K, phosphatidylinositol 3-kinase; PKA, cAMP-dependent protein kinase; PKB, protein kinase B; P-, dephosphorylation; RXR, retinoid X receptor; SREBP1c, sterol regulatory element–binding protein 1c; T, transformation; TGF, transforming growth factor; TR, transcription regulation; +P, phosphorylation; Z, catalysis; GPCR, G protein-coupled receptor; 15d-PGJ2, deoxy-delta prostaglandin J2; PDK/PDK1, 3-phosphoinositide-dependent protein kinase -1; ACACA, acetyl-CoA carboxylase; ACSL, acyl-CoA synthetase long-chain family members; ACLY, ATP citrate lyase; BCAA, branch chain amino acid; CISY, citrate synthase; DAG, diacylglycerol; ELOVL, elongation-of-very-long-chain-fatty acids; EMT, epithelial-mesenchymal transition; BEH, ethylene-bridged hybrid; 4E-BP1, eukaryotic translation initiation factor 4E binding protein 1; FADS1, fatty acid desaturase 1; FASN, fatty acid synthase; GSK3β, glycogen synthase kinase 3; GNAS, G protein αs- dependent adenylate cyclase; GRB2, growth factor receptor-bound protein 2; H-Ras, Harvey rat sarcoma viral oncogene homolog; HGF, hepatocyte growth factor; HXK, hexokinase; HSS, high-strength silica; HODE, hydroxyoctadecadienoic acid; INSIG2, insulin-induced gene 2; IRS-1 and IRS-2, insulin receptor substrates-1 and -2; TRIP, mediator complex subunit 1; MEK/MAP1, mitogen-activated protein kinase kinase 1; NCOA1, nuclear receptor coactivator 1; NRC1/SRC1, nuclear receptor coactivator 1; N-CoR, nuclear receptor corepressor; SMRT, nuclear receptor corepressors; NUDT1, nudix (nucleoside diphosphate-linked moiety X)-type motif 1; PtdIns(3,4,5)P3, phosphatidylinositol 3,4,5-triphosphate; P13K, phospatidylinositol 3-kinase; PtdIns(4,5)P2, phosphatidylinositol 4,5-biphosphate; PGE, prostaglandin; PTGIS, prostaglandin I2 (prostacyclin) synthase; PDGHS, prostaglandin-endoperoxide synthase 2 prostaglandin G/H synthase; COX2, cyclooxygenase 2; PDHA, pyruvate dehydrogenase (lipoamide) α1; QCs, quality controls; RARs, retinoic acid receptors; RXRA, retinoid X nuclear receptor (α; SHC, Src homology 2 domain containing transforming protein 1; SHP, small heterodimer partner; SOS, son of sevenless protein homologs 1 and 2; c-Raf-1, gene homolog 1; XIAP, X-linked inhibitor of apoptosis.
FIG. 4.
FIG. 4.
Integration of the metabolomics with transcriptomics data and their superimposition to build metabolic networks. A: Metabolic network of PPAR transcription pathway, which is connected to other metabolic processes such as lipid homeostasis, glucose, fatty acid metabolism, and inflammatory response. B: Network model of downstream of insulin-signaling pathways. The metabolites and the gene names shown in red are upregulated, and the same shown in blue are downregulated during insulin deficiency. B, binding; C, cleavage; CoA, coenzyme A; Erk, extracellular signal–related kinase; HPODE, hydroperoxylinoleic acid; IE, influence on expression; MAP, mitogen-activated protein; MAPK, MAP kinase; PDGF, platelet-derived growth factor; PI3K, phosphatidylinositol 3-kinase; PKA, cAMP-dependent protein kinase; PKB, protein kinase B; P-, dephosphorylation; RXR, retinoid X receptor; SREBP1c, sterol regulatory element–binding protein 1c; T, transformation; TGF, transforming growth factor; TR, transcription regulation; +P, phosphorylation; Z, catalysis; GPCR, G protein-coupled receptor; 15d-PGJ2, deoxy-delta prostaglandin J2; PDK/PDK1, 3-phosphoinositide-dependent protein kinase -1; ACACA, acetyl-CoA carboxylase; ACSL, acyl-CoA synthetase long-chain family members; ACLY, ATP citrate lyase; BCAA, branch chain amino acid; CISY, citrate synthase; DAG, diacylglycerol; ELOVL, elongation-of-very-long-chain-fatty acids; EMT, epithelial-mesenchymal transition; BEH, ethylene-bridged hybrid; 4E-BP1, eukaryotic translation initiation factor 4E binding protein 1; FADS1, fatty acid desaturase 1; FASN, fatty acid synthase; GSK3β, glycogen synthase kinase 3; GNAS, G protein αs- dependent adenylate cyclase; GRB2, growth factor receptor-bound protein 2; H-Ras, Harvey rat sarcoma viral oncogene homolog; HGF, hepatocyte growth factor; HXK, hexokinase; HSS, high-strength silica; HODE, hydroxyoctadecadienoic acid; INSIG2, insulin-induced gene 2; IRS-1 and IRS-2, insulin receptor substrates-1 and -2; TRIP, mediator complex subunit 1; MEK/MAP1, mitogen-activated protein kinase kinase 1; NCOA1, nuclear receptor coactivator 1; NRC1/SRC1, nuclear receptor coactivator 1; N-CoR, nuclear receptor corepressor; SMRT, nuclear receptor corepressors; NUDT1, nudix (nucleoside diphosphate-linked moiety X)-type motif 1; PtdIns(3,4,5)P3, phosphatidylinositol 3,4,5-triphosphate; P13K, phospatidylinositol 3-kinase; PtdIns(4,5)P2, phosphatidylinositol 4,5-biphosphate; PGE, prostaglandin; PTGIS, prostaglandin I2 (prostacyclin) synthase; PDGHS, prostaglandin-endoperoxide synthase 2 prostaglandin G/H synthase; COX2, cyclooxygenase 2; PDHA, pyruvate dehydrogenase (lipoamide) α1; QCs, quality controls; RARs, retinoic acid receptors; RXRA, retinoid X nuclear receptor (α; SHC, Src homology 2 domain containing transforming protein 1; SHP, small heterodimer partner; SOS, son of sevenless protein homologs 1 and 2; c-Raf-1, gene homolog 1; XIAP, X-linked inhibitor of apoptosis.

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