Nagasawa K et al. (2013), Significant modulation of the hepatic proteome ...

XB-ART-47831

Biol Open 2013 Aug 21;210:1057-69. doi: 10.1242/bio.20136106.

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Significant modulation of the hepatic proteome induced by exposure to low temperature in Xenopus laevis.

Nagasawa K , Tanizaki Y , Okui T , Watarai A , Ueda S , Kato T .

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The African clawed frog, Xenopus laevis, is an ectothermic vertebrate that can survive at low environmental temperatures. To gain insight into the molecular events induced by low body temperature, liver proteins were evaluated at the standard laboratory rearing temperature (22°C, control) and a low environmental temperature (5°C, cold exposure). Using nano-flow liquid chromatography coupled with tandem mass spectrometry, we identified 58 proteins that differed in abundance. A subsequent Gene Ontology analysis revealed that the tyrosine and phenylalanine catabolic processes were modulated by cold exposure, which resulted in decreases in hepatic tyrosine and phenylalanine, respectively. Similarly, levels of pyruvate kinase and enolase, which are involved in glycolysis and glycogen synthesis, were also decreased, whereas levels of glycogen phosphorylase, which participates in glycogenolysis, were increased. Therefore, we measured metabolites in the respective pathways and found that levels of hepatic glycogen and glucose were decreased. Although the liver was under oxidative stress because of iron accumulation caused by hepatic erythrocyte destruction, the hepatic NADPH/NADP ratio was not changed. Thus, glycogen is probably utilized mainly for NADPH supply rather than for energy or glucose production. In conclusion, X. laevis responds to low body temperature by modulating its hepatic proteome, which results in altered carbohydrate metabolism.

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Species referenced: Xenopus laevis
Genes referenced: acss2.2 agl aldob ces2.8 cs fubp1 g6pd gak gbe1 gck gk gnpda1 gpd1 grhpr.1 gys1 ids pfkm pklr pkm prkg1 pygl tkt tpi1

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	Fig. 1. Transitions in environmental temperature and hepatic proteins.(A) Relationship between cold-exposure time and water temperature. A plastic tank containing 1âl of 22Â°C water was transferred to an incubator at 5Â°C and allowed to cool. During that time, the temperature of the water was measured. (B) Image of SDS-PAGE gels used to separate X. laevis liver protein samples; 10âÂµg of each sample from either the control or cold-exposure group (three frogs each) was separated on 12% SDS polyacrylamide gels. Protein bands were visualized by staining with Coomassie brilliant blue R-250. Reducing (right panel) and non-reducing (left panel) conditions are shown.
	Fig. 2. Schematic view of sample and data processing.Each test sample, i.e. a protein extract mixture derived from three frogs prepared by bead crusher, was subjected to tryptic digestion. Subsequent nanoLCâMS/MS analysis was repeated three times. Automatic peak extraction and MASCOT MS/MS ion search were performed using Xome software. The triplicate data were processed individually in all combinations to calculate the protein ratios by non-label quantification using Mass Navigator software then protein ratios were averaged after normalization. The details are explained in the Materials and Methods and in Tableâ1.
	Fig. 3. Outlines of MASCOT MS/MS ions search. (A) Summary of MASCOT peptide identification in each search. aPeptide matches above identity threshold (P<0.05); bFDR â=â decoy hits/NCBInr hits. (B) Venn diagrams of identified proteins in individual searches. Proteins identified in at least two of the triplicate runs are highlighted in grey. (C) Comparison of valid identified proteins differentially expressed between the control and cold-exposure conditions. Proteins identified at least twice were compared (126 proteins from the control group and 100 proteins from the cold-exposure group); 81 proteins overlapped between both groups.
	Fig. 4. GO and pathway analyses of X. laevis liver proteomes.(A) Schematic view of the data processing procedure. The GI accessions of X. laevis proteins were converted to human RefSeq protein IDs by using NCBI HomoloGene and BLASTp, and then GO and pathway analyses were performed by using the DAVID program. FDR-corrected P values were defined by modified Fisher's exact test with the Benjamini and Hochberg FDR correction. The significantly identified GO:BP terms appearing deepest in the hierarchy and the significantly identified KEGG pathways are shown (FDR-corrected P<0.01). The details are explained in the materials and methods. (B,C) Comparison of enriched GO:BP terms (B) and KEGG pathways (C) in the list of proteins identified between the control and cold-exposure conditions.
	Fig. 5. Distribution of fold change in protein abundance.The bar chart shows protein ratios between the cold-exposure and control conditions for all 81 relatively quantified proteins.
	Fig. 6. Cold-exposure-induced changes in protein abundance associated with carbohydrate metabolism.Substrates and enzymes are as follows: (glycolysis/glyconeogenesis) G6P, glucose 6-phosphate; F6P, fructose 6-phosphate; FBP, fructose 1,6-bisphosphate; GAP, glyceraldehyde 3-phosphate; DHAP, dihydroxyacetone phosphate; 1,3BPG, 1,3-bisphosphoglyceric acid; 3PGA, 3-phosphoglyceric acid; 2PGA, 2-phosphoglyceric acid; PEP, Phosphoenolpyruvate; GCK, glucokinase; G6Pase, glucose-6-phaosphatase; GPI, glucose-6-phosphate isomerase; PFK, phosphofructokinase; FBPase, fructose-1,6-bisphosphatase; FBA, fructose 1,6-bisphosphate aldorase; TPI, triosephosphate isomerase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PGK, phosphoglyceric acid kinase; ENO, enolase; PEPCK, phosphoenolpyruvate carboxykinase; PK, pyruvate kinase; MDH, malate dehydrogenase; LDH, lactate dehydrogenase; ACL, ATP-citrate synthase (ATP-citrate lyase); ACS, acetyl-CoA synthase; ALDH, aldehyde dehydrogenase; ADH, alcohol dehydrogenase; (pentose phosphate pathway) 6PG, 6-phosphogluconate; Ro5P, ribose 5-phosphate; Ru5P, ribulose 5-phosphate; Xu5P, xylulose 5-phosphate; S7P, sedoheptulose 7-phosphate; E4P, erythrose 4-phosphate; G6PDH, glucose-6-phosphate dehydrogenase; 6PGDH, 6-phosphogluconate dehydrogenase; TKET, transketolase; TALD, transaldolase; (glycogen metabolism) G1P, glucose 1-phosphate; UDPG, uridine diphosphate glucose; PGM, phosphoglucomutase; UGPase, UDP-glucose pyrophosphorylase; GYS, glycogen synthase; PYGL, glycogen phosphorylase; GBE, glycogen branching enzyme; GDE, glycogen debranching enzyme; (others) Glycerol-3-P, glycerol 3- phosphate; F1P, fructose 1-phosphate; GK, glycerol kinase; G3Pase, glycerol-3-phosphatase; GPDH, glycerol-3-phosphate dehydrogenase; FK, fructokinase; ALDB, fructose-bisphosphate aldolase B; GAK, glyceraldehyde kinase; GRHPR, glyoxylate reductase/hydroxypyruvate reductase; TMABADH, 4-trimethylaminobutyraldehyde dehydrogenase; ACAT, acetyl-CoA acetyltransferase; mMDH, mitochondrial MDH; IDH, isocitrate dehydrogenase. Modified from portions of KEGG pathway map for â€˜glycolysis/gluconeogenesisâ€™ (00010) and â€˜Pentose phosphate pathwayâ€™ (00030).
	Fig. 7. Comparisons of free aromatic amino acid levels and glycogen levels in the liver between the control and cold-exposure conditions.(A) Free amino acids in the liver (nâ=â3). (B) Glycogen in the liver (nâ=â4). Each bar represents the s.e.m. *P<0.05 by Student's t-test.
	Fig. 8. Comparisons of glucose, glycerol, NADP, and NADPH levels in the liver and/or plasma between the control and cold-exposure conditions.(A,B) Glucose levels in the liver (nâ=â3) and plasma (nâ=â5). (C,D) NADP and NADPH levels in the liver (nâ=â4). (E,F) Glycerol levels in the liver (nâ=â4) and plasma (nâ=â4). Each bar represents the s.e.m. *P<0.05 by Student's t-test.
	Fig. 9. Schematic models of the early response to cold exposure in the liver of X. laevis.In cold exposure, hepatic glycogen and glucose are thought to be used in the pentose phosphate pathway for NADPH supply rather than in energy production through glycolysis. This mechanism suppresses oxidative stress derived from iron accumulation caused by hepatic erythrocyte degradation. Abbreviations for the substrate and enzymes are shown in Fig.â6.

References [+] :

Aizawa, Expression of erythropoietin receptor-like molecule in Xenopus laevis and erythrocytopenia upon administration of its recombinant soluble form. 2005, Pubmed, Xenbase