Article pubs.acs.org/jpr
Global Liver Proteome Analysis Using iTRAQ Reveals AMPK−mTOR− Autophagy Signaling Is Altered by Intrauterine Growth Restriction in Newborn Piglets Baisheng Long,†,‡,¶ Cong Yin,†,‡,¶ Qiwen Fan,†,‡ Guokai Yan,†,‡ Zhichang Wang,†,‡ Xiuzhi Li,†,‡ Changqing Chen,†,‡ Xingya Yang,†,‡ Lu Liu,†,‡ Zilong Zheng,†,‡ Min Shi,†,‡ and Xianghua Yan*,†,‡ †
College of Animal Sciences and Technology, Huazhong Agricultural University, Wuhan, 430070 Hubei, China The Cooperative Innovation Center for Sustainable Pig Production, Wuhan 430070, Hubei, China
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S Supporting Information *
ABSTRACT: Intrauterine growth restriction (IUGR) impairs fetal growth and development, perturbs nutrient metabolism, and increases the risk of developing diseases in postnatal life. However, the underlying mechanisms by which IUGR affects fetal liver development and metabolism remain incompletely understood. Here, we applied a high-throughput proteomics approach and biochemical analysis to investigate the impact of IUGR on the liver of newborn piglets. As a result, we identified 78 differentially expressed proteins in the three biological replicates, including 31 significantly up-regulated proteins and 47 significantly downregulated proteins. Among them, a majority of differentially expressed proteins were related to nutrient metabolism and mitochondrial function. Additionally, many significantly down-regulated proteins participated in the mTOR signaling pathway and the phagosome maturation signaling pathway. Further analysis suggested that glucose concentration and hepatic glycogen storage were both reduced in IUGR newborn piglets, which may contribute to AMPK activation and mTORC1 inhibition. However, AMPK activation and mTORC1 inhibition failed to induce autophagy in the liver of IUGR neonatal pigs. A possible reason is that PP2Ac, a potential candidate in autophagy regulation, is significantly down-regulated in the liver of IUGR newborn piglets. These findings may provide implications for preventing and treating IUGR in human beings and domestic animals. KEYWORDS: proteomics, AMPK, mTOR, autophagy, IUGR, PP2Ac, piglets
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mitochondrial function and antioxidant capacity,7 and altered nutrient metabolism.8 Additionally, IUGR increases the risk of developing obesity and diabetes in postnatal life. Perhaps IUGR increases hepatic gluconeogenic capacity and impairs β-cell function, ultimately leading to insulin resistance.9,10 However, the underlying mechanisms by which IUGR affects the growth, development, metabolism, and diseases of the liver in mammals remain poorly understood. AMP-activated protein kinase (AMPK), which can be activated by elevation of the AMP/ATP ratio, is a master regulator of energy balance.11 When cellular energy level is limited, AMPK can inhibit mammalian target of rapamycin
INTRODUCTION Intrauterine growth restriction (IUGR) is a serious problem in both human beings and domestic animals due to its high morbidity and mortality, low efficiency of feed utilization, and permanent adverse effects on postnatal life.1,2 Approximately 5− 10% of human infants worldwide and 15−20% of newborn piglets3,4 with low birth weight are termed as IUGR, which can be caused by multiple factors, such as maternal malnutrition, placental insufficiency, and environmental factors.2,3 Liver is a major organ of nutrient absorption and metabolism, whereas IUGR can impair hepatic growth and development during pregnancy.5 The weight of liver is reduced in IUGR fetuses compared with normal weight (NW) fetuses.4 Besides, IUGR fetal livers are accompanied by metabolic disorders in postnatal life, such as reduced oxidative phosphorylation,6 impaired © XXXX American Chemical Society
Received: January 1, 2016
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DOI: 10.1021/acs.jproteome.6b00001 J. Proteome Res. XXXX, XXX, XXX−XXX
Journal of Proteome Research
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complex 1 (mTORC1) and promote autophagy.12 The mTORC1 signaling pathway plays a vital role in regulating organismal growth and homeostasis via sensing and integrating environmental cues.13 Autophagy functions in clearance of degenerated proteins and organelles as well as nutrient recycling and energy production in response to nutrient starvation.14 Additionally, AMPK activation can also promote the processes of ATP generation, such as fatty acid oxidation and glucose uptake as well as mitochondrial biogenesis, which can increase oxidative catabolism of both glucose and fatty acids.15,16 A previous study has reported that IUGR can reduce glucose uptake from placenta and insulin concentration and increased hepatic gluconeogenic capacity, which may lead to insulin resistance and diabetes.9 This study also showed that hepatic mTOR activity levels were similar between those in NW and IUGR fetal sheep.9 In contrast, mTOR phosphorylation was markedly decreased in the small intestine mucosa and fetal liver.17,18 However, it is yet unknown whether autophagy is affected by IUGR in the liver of neonatal fetuses. Interestingly, recent studies have demonstrated that defective hepatic autophagy was closely linked to obesity, insulin resistance,19 and embryo developmental disorders.20 Therefore, IUGR stunts embryo development during pregnancy and increases the risk of developing diseases (e.g., obesity and diabetes) in postnatal life, which may be associated with autophagy deficiency. Protein phosphatase 2A (PP2A) is a highly conserved serine and threonine phosphatase with a functional role in multiple biological processes, including DNA replication, RNA splicing, translation, apoptosis, and stress responses.21,22 PP2A consists of a scaffold A subunit, a regulatory B subunit, and a catalytic C subunit.21 The catalytic C subunit contains two isoforms, PP2ACα and PP2ACβ, which share 97% of amino acid sequence identity.23 PP2ACα, the predominant isoform of catalytic C subunit, is more abundant and indispensable than PP2ACβ.24 Deletion of PP2ACα leads to early embryonic lethality at 6.5 days of embryonic development (E6.5), developmental defects, and fetal liver erythropoiesis retardation.24,25 Likewise, inhibition of PP2Ac by okadaic acid (OA) can stimulate AMPK and suppress autophagy in rat hepatocytes and cortical neurons.26−28 Paradoxically, the stimulative function of AMPK in autophagy is well-established.12 Additionally, PP2A inhibition reduces glucose production and glycogen storage, accompanied by insulin resistance.29 A possible explanation is that PP2A is required for glycogen storage. However, whether PP2A has an essential role in regulating the growth, development, metabolism, and diseases of IUGR fetuses is largely unknown. In this study, we applied a high-throughput proteomics approach to systematically investigate the distinction of liver proteome between IUGR and NW newborn piglets that are used as the animal model for studying human physiology and metabolism. As a result, 78 differentially expressed proteins were identified in the three biological replicates. Bioinformatics analysis revealed that these differentially expressed proteins participated in nutrient metabolism and tissue growth and development. Further analysis suggested that the AMPK− mTOR−autophagy signaling was altered by IUGR in newborn piglets. Our results may achieve a comprehensive understanding of the liver proteome altered by IUGR and potential mechanisms of IUGR formation. These findings may provide implications for preventing and treating IUGR in human beings and domestic animals.
Article
MATERIALS AND METHODS
Pigs and Tissue Samples
After term birth (day 114 of gestation), one IUGR newborn piglet and one NW newborn piglet were obtained from each of 7 gilts (n = 7). A piglet with a birth weight of less than 1.1 kg was randomly selected as IUGR from each litter, and a weight similar to average value was randomly selected as NW. Animal maintenance and experimental treatments were conducted in accordance with the ethical guidelines for animal research established and approved by the institutional Animal Care and Use Committee at Huazhong Agricultural University. Liver tissue samples for iTRAQ and Western blotting assays were treated by liquid nitrogen and then stored at −80 °C. The liver tissue samples for hematoxylin and eosin (H & E) staining and periodic acid−Schiff (PAS) staining were fixed in 4% paraformaldehyde for 24 h at room temperature and then processed for paraffin embedding. Liver tissue samples for transmission electron microscopy assessment were fixed in 0.1 M sodium cacodylate buffered (pH 7.4) 2.5% glutaraldeyde solution. It is noteworthy that all of the tissue samples were collected from a similar area on each organ. Additionally, the umbilical vein blood was collected and centrifuged at 3000g for 10 min at 4 °C, and then plasma was immediately stored in 500 μL aliquots at 4 °C for glucose analysis. Protein Preparation, Digestion, and iTRAQ Labeling
The liver samples from three IUGR and three NW fetuses were used for protein extraction. Liver tissues were milled to powder in mortar with lipid nitrogen. Subsequently, 150 mg of powder from each sample was mixed with 1 mL of lysis buffer containing 50 mM Tris (pH 8.8), 150 mM NaCl, 1 mM EDTA, and 1% Triton X-100 supplemented with protease inhibitors (1 μg/mL leupeptin, 2 μg/mL aprotinin, 1 μg/mL pepstatin A, and 100 μg/ mL PMSF) in a glass homogenizer. Homogenates were incubated on the ice for 20 min and then centrifuged at 12000g for 15 min at 4 °C. Finally, the supernatants were kept at −80 °C for iTRAQ and Western blotting assays. The supernatant containing precisely 100 μg protein of each sample was digested with Trypsin Gold (Promega, Madison, WI) at 37 °C for 16 h. Subsequently, peptides were dried by vacuum centrifugation and reconstituted in 0.5 M TEAB (Applied Biosystems, Milan, Italy). The tryptic peptides were labeled with isobaric iTRAQ tags (NW: 113, 114, 115; IUGR: 118, 119, 121). The labeled peptides were incubated for 2 h at room temperature and then mixed and dried by vacuum centrifugation. LC−MS/MS Analysis
Strong cationic-exchange chromatography (SCX) was performed on a LC-20AB HPLC Pump system (Shimadzu, Kyoto, Japan) to separate samples. The labeled peptide mixtures were eluted with 4 mL of buffer A (25 mM NaH2PO4 in 25% ACN, pH 2.7) at a flow rate of 1 mL/min for 10 min, and then a gradient elution with 5−60% and 60−100% buffer B (25 mM NaH2PO4, 1 M KCl in 25% ACN, pH 2.7) for 27 and 1 min, respectively, was performed. The elution process was monitored by measuring the absorbance at 214 nm. A total of 20 fractions were collected every 1 min, desalted with a Strata X C18 column (Phenomenex), and vacuum-dried. Subsequently, each fraction was centrifuged at 20000g for 10 min after resuspending in buffer A (2% ACN, 0.1% FA). Samples of 5 μg of labeled peptides were loaded onto a LC20AD nano-HPLC (Shimadzu, Kyoto, Japan) by the autosampler onto a 2 cm C18 trap column with a flow rate of 8 μL/min for 4 min. The peptides were then eluted onto a 10 cm analytical B
DOI: 10.1021/acs.jproteome.6b00001 J. Proteome Res. XXXX, XXX, XXX−XXX
Article
Journal of Proteome Research
Figure 1. Characteristics of bodies and livers in NW and IUGR newborn piglets. (A) The phenotype (left) and birth weight (right) of NW and IUGR fetuses. (B) The phenotype (left) and weight (right) of NW and IUGR fetal livers. (C) Analysis of liver mass to body mass ratio between IUGR and NW fetuses. Values are means ± SEM *, p < 0.05; ***, p < 0.001 (n = 7). (D) The hepatic morphology of NW and IUGR neonates was observed using hematoxylin and eosin (H & E) staining. Scale bars represent 100 μm (above) and 50 μm (below).
C18 column (inner diameter 75 μm) with a 44 min linear gradient run at a flow rate of 300 nL/min was from 2% to 35% buffer B (95% ACN, 0.1% FA), followed by 2 min linear gradient to 80% buffer B and maintenance at 80% buffer B for 4 min, finally returning to 5% buffer B in 1 min. After liquid-phase separation, the peptides were subjected to nanoelectrospray ionization follow by tandem mass spectrometry (MS/MS) in QEXACTIVE (Thermo Fisher Scientific, San Jose, CA) coupled online to the HPLC. Orbitrap detected intact peptides with a resolution of 70 000 and a mass range of 350−2000 m/z. MS/MS analysis were recorded with a resolution of 17 500 and a mass range of 100−1800 m/z. MS/MS analysis was required for the 15 most abundant precursor ions, which were above a threshold ion count of 20 000 in the MS survey scan, including a following dynamic exclusion duration of 15 s.
mass spectrometry proteomics data (raw data, search, and peak list file) have been deposited to the ProteomeXchange Consortium30 via the PRODE partner repository with the data set identifier PXD003524. A quantitative protein with a ratio of >1.2 or 1.2 or
