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Intrauterine growth restriction programs the hypothalamus of adult male rats: integrated analysis of proteomic and metabolomic data Amanda Paula Pedroso, Adriana Pereira de Souza, Ana Paula Segantine Dornellas, Lila Missae Oyama, Cláudia Maria Oller Nascimento, Gianni Mara Silva Santos, José C. Rosa, Ricardo Pimenta Bertolla, Jelena Klawitter, Uwe Christians, Alexandre Keiji Tashima, and Eliane Beraldi Ribeiro J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.6b00923 • Publication Date (Web): 17 Mar 2017 Downloaded from http://pubs.acs.org on March 19, 2017

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Journal of Proteome Research

Intrauterine growth restriction programs the hypothalamus of adult male rats: integrated analysis of proteomic and metabolomic data

Amanda P. Pedroso1, Adriana P. Souza1, Ana P. S. Dornellas1, Lila M. Oyama1, Cláudia M. O. Nascimento1, Gianni M. S. Santos2, José C. Rosa3, Ricardo P. Bertolla4, Jelena Klawitter5, Uwe Christians5, Alexandre K. Tashima6*, Eliane B. Ribeiro1*

1

Department of Physiology, Universidade Federal de São Paulo UNIFESP, São Paulo, SP,

Brazil. 2

Division of Applied Statistics, Universidade Federal de São Paulo UNIFESP, São Paulo, SP,

Brazil. 3

Protein Chemistry Center, Department of Molecular and Cell Biology, Ribeirão Preto Medical

School, Universidade de São Paulo, Ribeirão Preto, SP, Brazil. 4

Department of Surgery, Universidade Federal de São Paulo UNIFESP, São Paulo, SP, Brazil.

5

iC42 Clinical Research and Development, Department of Anesthesiology, University of

Colorado Denver, Anschutz Medical Campus, Aurora, CO, USA. 6

Department of Biochemistry, Universidade Federal de São Paulo UNIFESP, São Paulo, SP,

Brazil.

1 ACS Paragon Plus Environment


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*Corresponding authors: Alexandre Keiji Tashima Universidade Federal de São Paulo, Departamento de Bioquímica Rua Botucatu 862 Vila Clementino, 04023-062, São Paulo, SP, Brasil E-mail: [email protected] Phone/Fax: 55 11 5576-4848

Eliane Beraldi Ribeiro Universidade Federal de São Paulo, Departamento de Fisiologia Rua Botucatu 862 Vila Clementino, 04023-062, São Paulo, SP, Brasil E-mail: [email protected] Phone/Fax: 55 11 5576-4765


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Abstract Programming of hypothalamic functions regulating energy homeostasis may play a role in intrauterine growth restriction (IUGR)-induced adulthood obesity. The present study investigated the effects of IUGR on the hypothalamus proteome and metabolome of adult rats submitted to 50% protein-energy restriction throughout pregnancy. Proteomic and metabolomic analyzes were performed by data independent acquisition mass spectrometry and multiple reaction monitoring, respectively. At age 4 months, the restricted rats showed elevated adiposity, increased leptin and signs of insulin resistance. 1356 proteins were identified and 348 quantified while 127 metabolites were quantified. The restricted hypothalamus showed downregulation of 36 proteins and 5 metabolites and up-regulation of 21 proteins and 9 metabolites. Integrated pathway analysis of the proteomics and metabolomics data indicated impairment of hypothalamic glucose metabolism, increased flux through the hexosamine pathway, deregulation of TCA cycle and the respiratory chain, and alterations in glutathione metabolism. The data suggest IUGR modulation of energy metabolism and redox homeostasis in the hypothalamus of male adult rats. The present results indicated deleterious consequences of IUGR on hypothalamic pathways involved in pivotal physiological functions. These results provide guidance for future mechanistic studies assessing the role of intrauterine malnutrition in the development of metabolic diseases later in life.

Keywords: Protein-energy restriction, low birth weight, metabolic programming, obesity, hypothalamus, label-free proteomics, metabolomics, energy metabolism, redox homeostasis.



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Introduction Epidemiological and experimental data have indicated that intrauterine growth restriction (IUGR), triggered by placental dysfunction or inadequate maternal diet, may increase the risk of metabolic disorders in adulthood. According to the thrifty phenotype hypothesis1, malnutrition during intrauterine life induces metabolic and physiological adaptations that could permanently change structure and function of tissues, a process known as metabolic programming. Although these changes allow fetus survival under conditions of nutrient shortage, they could have deleterious effects when food supply is restored. Obesity, cardiovascular diseases and type 2 diabetes are commonly related to low birth weight2,3. Obesity results from the interaction of many factors, and the increase of its prevalence worldwide has been associated with a sedentary lifestyle and consumption of high-energy diets. In countries undergoing nutritional transition, the coexistence of malnutrition and overweight/obesity has highlighted the involvement of early malnutrition as a factor promoting obesity4-7. Several authors have related obesity development as a consequence of IUGR to changes in structure and function of the appetite regulatory centers in the brain8-9. The hypothalamus integrates multiple central and peripheral mechanisms that control energy homeostasis. It receives neural, hormonal and metabolic signals, which provide information on body energy condition and affect the production of anabolic or catabolic mediators. Disorders of these central mechanisms are associated with obesity10-12. Previous results of our group have shown impairment of the central actions of insulin13 and serotonin14, important hypothalamic anorexigenic mediators, in the adult offspring of rats undernourished during pregnancy. Resistance to leptin and sibutramin have also been shown15.


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The potential of proteomics and metabolomics as powerful tools for nutritional studies has been highlighted. Using two-dimensional gel electrophoresis (2-DE), we have recently shown that IUGR affected metabolic pathways in the adipose tissue of adult rats, favoring obesity development16. Maternal protein deprivation reportedly altered the expression of hypothalamic proteins related to energy-sensing pathways, redox status and amino acid metabolism, as well as impacted serum metabolites related to fatty-acid -oxidation and amino acid metabolism in rats17,18. Alterations of hypothalamic proteins related to hormonal secretion and synaptic remodeling in addition to changes in glutamatergic pathways in the prefrontal cortex of adult rats have also been shown19. The present study evaluated the long term effects of IUGR, induced by protein-energy restriction, on the hypothalamic proteome and metabolome of male rats. The integrated analysis of proteins and metabolites allowed the identification of pathways modulated by early malnutrition. These results indicate an impact of IUGR on mechanisms relevant to energy metabolism and redox homeostasis in the hypothalamus of male adult rats.



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Experimental Procedures

Animals, sample collection and serum analysis This study was approved by the Research Ethics Committee of the Universidade Federal de São Paulo – UNIFESP. Wistar rats (Rattus norvegicus) were kept under controlled conditions of light (12 h light:12 h dark cycle, lights on at 6 am) and temperature (22 ± 1°C) and had free access to tap water throughout the experimental period. The food provided to animals consisted of standard rat chow (Nuvilab CR-1, Sogorb Ltda, Brazil). Three-month-old female rats (4 – 5 subjects) were mated and the first day of pregnancy was determined by the presence of spermatozoa in vaginal smears. Pregnant rats were housed individually and randomly assigned to be a control or a restricted dam. The control dams were fed ad libitum throughout pregnancy. The restricted dams were fed the same diet but restricted to 50% of the intake of the control dams at the same pregnancy day. Dams from both groups received food ad libitum during lactation. On the delivery day, the pups were weighed and litter size was adjusted to eight per dam. Male offspring were housed four per cage and were fed ad libitum. Food intake and body weight were measured once a week from weaning until 4 months of age, when rats were sacrificed by decapitation. Trunk blood was collected and serum aliquots were stored at -80 oC until further analysis. White fat depots (retroperitoneal, mesenteric and gonadal) were dissected and weighed. The skull was rapidly opened, the hypothalamus was dissected having the thalamus as the dorsal limit, with rostral and caudal limits being the optic chiasm and the mammillary bodies, respectively20, and immediately frozen in liquid nitrogen.


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Glucose levels were determined by enzymatic colorimetric method using commercial kit (Labtest Diagnóstica, Lagoa Santa, Brazil). Leptin and insulin concentration was measured by Multiplex Assays using Luminex technology (Merck Millipore, Darmstadt, Germany). Homeostatic model assessment of insulin resistance (HOMA-IR) was calculated using the formula: fasting plasma glucose (mmol/L) x insulin (µU/mL)/22.521, 22.

Proteomic Analysis Protein extraction and digestion Each hypothalamus was homogenized in 1 mL of lysis buffer containing 8 M urea, 75 mM NaCl, 1 M Tris and complete Mini Protease Inhibitor Cocktail Tablets (Roche Diagnostics, Indianapolis, IN, USA), and centrifuged at 19,000 g for 30 minutes at 4 oC. Protein concentration in the supernatants was determined using 2-D Quant Kit (GE Healthcare, Waukesha, WI, USA) according to manufacturer’s recommendations. Aliquots of 800 g of protein were applied to Amicon Ultra-4 Centrifugal 3,000 NMWL filter devices (Merck Millipore) to perform buffer exchange to 50 mM ammonium bicarbonate to concentrate protein. This was followed by a second determination of proteins concentration in the recovered supernatants. Next, 25 L of 0.2% solution of RapiGest SF (Waters, Milford, MA, USA) were added to an aliquot of 200 g of protein and incubated at 80 oC for 15 min. Samples were reduced with 5 mM DTT at 60 oC for 30 min and then alkylated in the dark with 10 mM iodoacetamide at room temperature for 30 min. Proteins were digested using trypsin (Promega, Fitchburg, WI, USA) at a 1:100 (wt:wt) enzyme:protein ratio, at 37 oC overnight. Digestion was stopped by addition of 10 L of 5% trifluoroacetic acid and incubation at 37 oC for 90 min. Samples were centrifuged at 19,000 g for



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10 minutes at 4 oC and supernatants were recovered. The final concentration of protein was around 2 g/L. Mass Spectrometry (Data Independent Acquisition Mode) Digested samples (4 biological replicates per group, 3 technical replicates) were analyzed in a Synapt G2 HDMS Q-TOF mass spectrometer (Waters) coupled to a nanoAcquity UPLC chromatographic system. Samples were injected onto a trap column (nanoAquity C18 trap column Symmetry 180 m x 20 mm, Waters) and transferred by an elution gradient to an analytical column (nanoAcquity C18 BEH 75 µm x 150 mm, 1.7 mm, Waters). Buffer A (0.1% formic acid in water) and B (0.1% formic acid in acetonitrile) were used to generate a 7-35% B elution gradient run over 92 minutes at a flow rate of 275 nL/min. Data were acquired in HDMSE mode, switching from low (4 eV) to high (ramped from 19 to 45 eV) collision energy, for accurate measurement of both intact peptides and fragments. Glu-fibrinopeptide B (Waters) was infused using a nanoLockSpray apparatus and scanned every 30 s for external calibration. Data Analysis ProteinLynx Global Server software version 3.0.1 (Waters) was used for mass spectrometry data processing and also for database search against Rattus norvegicus sequences in the UniProtKB/Swiss-Prot database (www.uniprot.org, including 9485 entries). The following search parameters were used: carbamidomethylation of cysteines as fixed modification, oxidation of methionine, N-terminal acetylation, glutamine and asparagine deamidation as variable modifications, up to 2 missed cleavage sites were allowed for trypsin digestion and automatic fragment and peptide mass tolerance. The following criteria were set for protein identification: a minimum of 1 fragment ions per peptide, 5 fragment ions per protein and 2 peptides per protein, and the false discovery identification rate was set to 1%, estimated by a


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simultaneous search against a reversed database. Label-free quantitative assessments based on peptide intensities were performed. The integrated intensities of the three most intense peptides of each identified protein (Top 3 matched peptides) were used for relative quantitation23. Results were exported into Excel files. Proteins identified in at least 2 technical replicates and 3 biological replicates were included in the analysis. Additionally, proteins not detected in any of the 12 replicates of one group (indicating that the intensities were below the detection limit), but identified in at least 4 replicates in the other group were listed and included in the pathway analysis. Normalization was performed in each sample according to the sum of protein intensities.

Two-dimensional gel electrophoresis Two-dimensional (2-D) gels were performed as described previously24. Briefly, the 2-D gels (6 biological replicates per group) were stained with Coomassie Blue G-250, scanned in a GS-710 Calibrated Imaging Densitometer (Bio-Rad, Hercules, CA, USA) and analyzed using PDQuest Image Analysis Software (version 8.0, Bio-Rad). After automated spot detection, spot matching was performed manually. Normalization was performed using the “total quantity of valid spots” method of the software. The results were exported into Excel files. The protein identifications were based on the corresponding spots of our previous work of rat hypothalamus24.



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Metabolomic Analysis Metabolite extraction All procedures were performed according to the protocol published elsewhere25. Briefly, each hypothalamus (8 biological replicates per group) was homogenized in 500 L of 80% (v/v) cooled methanol, incubated during 4 hours at -80oC and centrifuged. The supernatant was transferred to a new tube and the pellet was mixed with 400 L of 80% (v/v) methanol, followed by 30 minutes incubation at -80oC. The supernatants from both extractions were combined and dried in a SpeedVac concentrator centrifuge (Thermo Fisher, Waltham, MA, USA). Regarding serum samples, aliquots of 200 L (8 biological replicates per group) were mixed with 800 L of cooled methanol. After incubation for 6 hours at -80oC, samples were centrifuged at 14,000 g for 10 minutes at 4 oC and dried in a SpeedVac. Samples were stored at -80oC until mass spectrometry analysis. Mass spectrometry (Selected Reaction Monitoring-based strategy) Dried samples were suspended in 20 L of 50 % (v/v) acetonitrile containing 0.2 mM of methionine-d3 as internal standard. Samples were analyzed in a single batch using an Agilent 1100 series HPLC system consisting of a G1312 binary pump, a G1322A vacuum degasser, and a G1316A thermostated column compartment (Agilent Technologies, Santa Clara, CA, USA). The HPLC system was interfaced with an ABSciex 5500 hybrid triple quadrupole mass spectrometer (Sciex, Concord, ON, Canada) operating with an electrospray ionization source (ESI) in positive/negative switch mode. Transitions were analyzed via selected reaction monitoring (SRM). The specific Q1>Q3 transitions for positive/negative ion switching were identical to the analytical platform of polar compounds previously described25, 26 . Eight


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microliters of the extracted sample were injected onto a 3.0 x 150mm 3μm Luna HILIC column (Phenomenex, Torrance, CA, USA). Data analysis Raw data were extracted using the MarkerViewTM v1.2.1 software (Sciex). Data extraction parameters were gaussian smooth width of 2 points, peak splitting factor of 5 points, one peak limited per chromatogram, a minimum intensity of 1000 cps, a minimum signal/noise of 5 and a minimum peak width of 5 points. Retention time correction was performed based on methionine-d3. It has been reported that when this method is performed the largest peak in the chromatogram is the correct peak for 80-90% of the targeted metabolites25. Herein, peaks were reviewed manually and only metabolites showing a single peak in the chromatogram were considered for relative quantification. Results tables were exported into Excel files. For hypothalamic samples, normalization was performed in each sample according to the sum of intensities of all quantified metabolites. For serum samples, values were normalized to methionine-d3.

Statistical analysis Body and white fat depots mass, food intake, leptin and HOMA-IR are expressed as mean ± SEM. Statistical analysis was performed in Statistica 12 Software (StatSoft, Tulsa, OK, USA). Significance of the differences between the restricted and the control group was determined using unpaired Student’s t-test considering p < 0.05 as significant.



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Pathway Analysis and Interaction Network Pathway analysis of significantly altered hypothalamic proteins and metabolites was performed using the Integrated Molecular Pathway Level Analysis – IMPaLA (http://impala.molgen.mpg.de/)27. Pathway over-representation analysis was performed according to the KEGG (Kyoto Encyclopedia of Genes and Genomes) database. P-values were corrected for multiple testing (FDR, Benjamini-Hochberg) and significance was set to q < 0.05. Search Tool for Interacting Chemicals – STITCH (http://stitch.embl.de/)28 was used to generate interaction network between altered proteins and metabolites with confidence score set at 0.7 (high confidence).

Results Body and fat depots mass, food intake and HOMA-IR At birth, body weight of the restricted group was significantly lower (18%) than that of the control group (Table 1). Since weaning, there were no differences in body weight and food intake between control and restricted groups, except in the fifth week, when the restricted offspring ate 30% less (p = 0.014) than controls (data not shown). At 4 months of age, body weights and food intake were similar between groups (Table 1). The mass of retroperitoneal and gonadal fat depots and the sum of the retroperitoneal, gonadal and mesenteric depots were significantly higher in the restricted than in the control group (Table 1). A significant increase of leptin levels was detected in restricted group. Moreover, the restricted rats showed a 68% non-significant (p = 0.064) increase in HOMA-IR (Table 1).


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Proteomics A total of 1356 proteins, each one identified by at least 2 peptides with 99% confidence, comprised the male rat hypothalamus dataset. After application of the inclusion criteria for quantification (presence in at least 2 technical replicates and 3 biological replicates), 318 proteins were compared between the groups (Table S1 – supporting information). Table S2 (supporting information) details MS information on the proteins affected by IUGR. Table 2 shows the proteins significantly affected by IUGR in the rat hypothalamus. Nineteen proteins were down-regulated while 8 proteins were up-regulated in the hypothalamus of the restricted group. Additionally, 17 proteins were identified only in the control group, while 13 proteins were identified only in the restricted group. It is interesting to point out that among the altered proteins, several participate in oxidative phosphorylation (ATP synthase subunit beta, cytochrome c oxidase subunit 5B, cytochrome c oxidase subunit 6C-2 and cytochrome c oxidase subunit 7A2 were down-regulated, and ATP synthase subunit alpha and V-type proton ATPase subunit B were up-regulated), in the TCA cycle (dihydrolipoyl dehydrogenase and 2oxoglutarate dehydrogenase, with increased and decreased levels, respectively) and in glycolysis (hexokinase-2). Moreover, glutathione synthetase, glutathione reductase and glutathione Stransferase omega-1, proteins that take part in the glutathione metabolism, were all up-regulated.

Two-dimensional gel-electrophoresis Technical validation of protein expression was carried out by 2-D gels of the hypothalamus of control and restricted rats, using spot identification data previously obtained24. Table 3 shows that 4 proteins significantly affected by IUGR, as analyzed by the present labelfree quantitative approach, were also present in the 2D gel. The two methods yielded similar



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results for the effect of IUGR on the expression of mitochondrial ATP synthase subunit alpha, fructose-bisphosphate aldolase A (both identified in two different spots in the 2D gel) and ubiquitin carboxyl-terminal hydrolase isozyme L1 (Figure S1 – supporting information). For alcohol dehydrogenase, however, a significantly increased expression was found by the labelfree approach but not by the 2D gel method.

Metabolomics The analysis of the hypothalamic and serum samples included, respectively, 127 and 115 different metabolites that met the inclusion criteria (presence of a single chromatographic peak) (Table S3 and Table S4 – supporting information). The restricted group had decreased levels of 5 metabolites and increased levels of 9 metabolites in the hypothalamus when compared to controls (table 4). Additionally, the results of serum metabolites showed reduced levels of 14 compounds while 5 metabolites had raised levels in the restricted group (table 5). Single reaction monitoring (SRM) transitions are shown on tables 4 and 5. Among the hypothalamic metabolites altered by IUGR, 3 participate in the TCA cycle (malate, GDP and FAD). The analysis of serum metabolites showed altered levels of compounds related to amino acids metabolism (glutamate, aspartate, pyruvate, glucosamine-6-phosphate, arginine, acetyl-L-ornithine), carbohydrate metabolism (aspartate, malate, pyruvate and 2,3diphosphoglyceric acid) and glutathione metabolism (glutamate and glutathione disulfide). Interestingly, three metabolites showed altered levels in both hypothalamus and serum: malate, glutamate and glucose.


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Pathway Analysis and Interaction Network Table 6 depicts the pathways that may have been significantly deregulated by IUGR in the hypothalamus, as indicated by the integrated pathway analysis. There were indications of altered glycolysis, TCA cycle, oxidative phosphorylation, and amino acids and glutathione metabolism. Interaction network of altered proteins and metabolites were significantly enriched (p = 1.28e-6) and corroborated pathway analysis (Figure S2 – supporting information).

Discussion Several studies point towards a relationship of obesity in adulthood and low birth weight, which may result from IUGR caused by altered maternal diet. This relationship has been associated with deregulation of appetite regulatory centers in the brain. The current study was designed to assess the effects of IUGR on the adult male hypothalamic proteome and metabolome, since this brain region plays a key role in regulating energy homeostasis. Additionally, serum metabolites were also analyzed. In the present study, a feeding protocol of 50% food restriction throughout pregnancy resulted in low birth weight offspring. Using the same protocol, several studies reported IUGR2930

. At 4-mo old, there were no significant differences between control and restricted groups

regarding body weight. However, the restricted group showed excess fat deposition, high leptin levels, and a trend to increased HOMA-IR, indicating impaired insulin sensitivity, characteristics related to obesity and diabetes. Available data about the effects of IUGR on metabolic characteristics in adulthood are divergent. We have previously shown that 50% food restriction during the first and second weeks of pregnancy resulted in increased adulthood body mass in the female but not in the male



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offspring13. Applying the same protocol, increased adiposity for both genders was reported by other authors31. Increased abdominal obesity with normal body mass was observed in males when a protocol of 70% food restriction during pregnancy was investigated32. We have recently demonstrated gender differences in the proteome of adipose tissue of rats submitted to IUGR: while in females the protein expression profile indicated established obesity at 4 months age, the male results showed a metabolic status favoring later obesity development16. Other reports have indicated that the impact of food restriction during intrauterine life in determining phenotypic characteristics in adulthood depends on several variables, such as timing, severity and type of restriction, post-natal growth rate and gender13, 33-35. Insulin resistance and glucose intolerance as effects of IUGR have been reported in both humans and animal models36-38. Men exposed to famine during fetal life had permanent changes in insulin-glucose metabolism39, 40. In a severe food restriction model, rats exhibited increased levels of leptin32, similar to the present findings. Rapid catch-up growth after birth can amplify such alterations32,41. Using an utero-placental insufficiency model of IUGR, it has been shown that male pups had dysregulated TNF- system and impaired glucose tolerance42. Taken together, our results showed restricted male rats at 4 months of age with elevated adiposity, increased levels of leptin and signs of insulin resistance. These findings of absence of hyperphagia with increased body adiposity confirm our previous results13, 14, 16 and may be related to the previously demonstrated low brown adipose tissue activity43, altered locomotor activity/energy expenditure44 and enhanced metabolic efficiency8. The main findings of the present study regarding the effects of IUGR on hypothalamic proteome and metabolome are summarized in figure 1 and discussed below.


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The mass spectrometry-based metabolomics data indicated significantly increased levels of glucose in both the hypothalamic tissue (23%) and the serum (12%) of the restricted group. Glucose is the main fuel to the brain cells and its concentration must be kept within physiological range. The hypothalamus has glucose sensing neurons and impairment of hypothalamic glucose signaling has been related to obesity and diabetes45. In rats with diet-induced obesity, we have demonstrated that abolition of the hypophagia caused by centrally administered glucose correlated with impairment of hypothalamic glucose sensing and signaling. Additionally, an excess glucose reached the hypothalamus of the DIO rats after a peripheral load46. High hypothalamic glucose concentration has also been reported in diabetic rats47. The hypothalamus also plays a role in glycemic control, which is influenced by hormones, such as insulin and leptin48. Here we found hyperleptinemia and signs of decreased peripheral insulin sensitivity, suggesting impairment of leptin’s and insulin’s actions. These findings agree with previous demonstrations that IUGR led to impaired insulin signaling in the adult rat hypothalamus13 and reduction of leptin transport to the brain49. Taken together, these data support the suggestion of deranged hypothalamic control of glucose metabolism by IUGR. Consistent with the increased hypothalamic levels of glucose, we also found increased levels of UDP-N-acetylglucosamine, the end-product of metabolism via fructose-6-phosphate in the hexosamine biosynthesis pathway. Increased flux in this pathway has been attributed an important role in the alterations induced by chronic hyperglycemia, namely increased mitochondrial ROS production, pro-inflammatory activation, cell damage, and insulin resistance50-52. The present findings indicate that the hypothalamus may also be subjected to these deleterious effects induced by glucose excess. Interestingly, it has recently been suggested that the post-translational addition of O-linked-N-acetylglucosamine (O-GlcNAc) to histone



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proteins, a mechanism mediating epigenetic influences on gene expression, could be a link between prenatal malnutrition and later development of obesity53. The present integrated analysis of proteins and metabolites indicated significant enrichment of the glycolytic pathway and the TCA cycle. Glucose metabolism through glycolysis and/or TCA cycle was reportedly decreased in the brain of obese Zucker rats54. Despite the increase in glucose levels in the hypothalamus, hexokinase-2 (one of the isoenzymes that phosphorylates glucose into glucose-6-phosphate in the first step of glycolysis) and fructosebisphosphate aldolase A (catalyses the cleavage of fructose-1,6-bisphosphate into glyceraldehyde 3-phosphate and dihydroxyacetone-phosphate), were down-regulated in the restricted group. Moreover, the oxidative decarboxylation of pyruvate to acetyl-CoA requires the pyruvate dehydrogenase complex. Two components of this complex, dihydrolipolyl dehydrogenase and FAD, showed decreased levels in the restricted group. Furthermore, these components are also part of the alpha-ketoglutarate dehydrogenase complex, that converts alpha-ketoglutarate to succinyl-CoA in the TCA cycle. This complex also had increased levels of alpha-ketoglutarate dehydrogenase. Interestingly, it has been shown that the alpha-ketoglutarate dehydrogenase complex is sensitive to reactive oxygen species (ROS), is also able to generate ROS, and its impaired function has been associated with neurodegenerative diseases55. Moreover, it is important to point out that malate levels were decreased. All these observed alterations indicate an imbalance in glycolysis, in pyruvate transformation to acetyl-CoA, and in the TCA cycle. In the present study, both circulating and hypothalamic levels of the excitatory neurotransmitter glutamate were decreased. Glutamate can be synthetized in neurons by amination of TCA-derived alpha-ketoglutarate and is also produced in neurons and astrocytes in the glutamate/GABA-glutamine cycle56. Impairment of this cycle has been reported in obesity


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and diabetes models in several brain regions54. Proteomic analysis of cortical and hypothalamic tissue of rats submitted to intrauterine protein restriction evidenced changes in synaptic transmission and glutamate metabolism19. Increased GABA production from glutamate, due to a single-nucleotide polymorphism of the GAD2 gene leading to increased glutamate decarboxylase 2 activity, has been associated with low birth weight and obesity in children57. Glutamate has been shown to either increase or decrease food intake. When injected into the lateral hypothalamus, the neurotransmitter stimulated feeding in normal satiated rats58. When injected into the lateral cerebral ventricle, glutamate decreased food intake and spontaneous activity in both lean and obese rats, acting through various mediators of food intake59. Besides its role as a neurotransmitter, glutamate is substrate for the synthesis of proteins and other nitrogen-containing molecules and also plays a relevant role as a regulator of metabolic pathways. Through the malate/aspartate shuttle, which allows mitochondrial oxidation of glycolysis-derived NADH in the respiratory chain, glutamate and malate play a relevant role in glucose utilization in neurons60, 61. Regarding the respiratory chain electron transport, it is important to highlight that three subunits of cytochrome c oxidase (complex IV) and ATP synthase subunit beta were downregulated while ATP synthase subunit alpha was up-regulated in the hypothalamus of the restricted group. This suggests imbalance in the production of energy in the form of ATP. Disturbances of mitochondrial respiratory chain could indicate mitochondrial dysfunction, which has been linked with obesity development and diabetes62, 63. Glutathione (GSH), a tripeptide consisting of amino acids glutamate, cysteine and glycine, neutralizes reactive oxygen species during its oxidation to glutathione disulfide (GSSG), which is reduced by glutathione reductase. In the present study, the expression of the enzymes



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glutathione synthetase, glutathione reductase and glutathione S-transferase omega-1 (involved in detox processes) were up-regulated in the hypothalamus of the restricted rats. These data suggest an impairment of this antioxidant defense system. Derranged glutathione metabolism has been associated with neurodegenerative and metabolic diseases64, 65. Disorders in the redox system were also detected in the hypothalamus of neonatal rats subjected to protein restriction during pregnancy17. In conclusion, the present results indicated that IUGR caused impairment of hypothalamic glucose metabolism, increased flux through the hexosamine pathway, deregulation of respiratory chain and alterations in glutathione metabolism. Taken together, the data suggest IUGR modulation of energy metabolism and redox homeostasis in the hypothalamus of male adult rats. The present results thus indicated deleterious consequences of IUGR on hypothalamic pathways involved in pivotal physiological functions. These pieces of information provide important insights to direct future work to clarify the molecular mechanisms programmed by the exposure to poor nutrient supply during the intrauterine life. Further experiments are warranted to ascertain the mechanisms undergoing the hypothalamic response to IUGR.


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Supporting Information The following files are available free of charge on the ACS Publications website http://pubs.acs.org: Figure S1: Representative image of a two-dimensional gel of hypothalamic proteins of a control male showing proteins analyzed by either shotgun proteomics or twodimensional gel electrophoresis; Figure S2: Interaction network of hypothalamic proteins and metabolites altered in the restricted group; Table S1: List of all quantified proteins in the hypothalamus of control and restricted rats; Table S2: Mass spectrometry information of the proteins significantly affected by intra-uterine growth restriction in the adult male rat hypothalamus; Table S3: List of all quantified metabolites analyzed in the hypothalamus of control and restricted rats; Table S4: List of all quantified metabolites analyzed in the serum of control and restricted rats.

Conflict of interest disclosure The authors declare no competing financial interest.

Acknowledgement We thank Dr. Josias F. Pagotto for the support in mass spectrometric analysis of proteomics data. We thank Dr. Douglas Andrade and Dr. Helio Martins (Sciex Brazil) for technical support. This work was supported by the Brazilian agencies CNPq (Grant No. 478550/2009-0) and FAPESP (Grant No. 2012/19321-9 to AKT and 2010/20268-0 to EBR).



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Figure legend Figure 1: Main effects of intrauterine growth restriction (IUGR) on hypothalamic proteome and metabolome of adult male rats. Proteins/metabolites in blue and red represent down and upregulation, respectively. Hk2: hexokinase-2; Aldoa: Fructose-bisphosphate aldolase A; PDC: pyruvate dehydrogenase complex; Pdh: pyruvate dehydrogenase; Dlat:dihydrolipoamide acetyltransferase; Dld: Dihydrolipoyl dehydrogenase; Ac-CoA: acetyl-CoA; α-KG: alfaketoglutarate; AKDC = α-ketoglutarate dehydrogenase complex; Ogdh: alpha-ketoglutarate dehydrogenase; Dlst: dihydrolipoyl succinyltransferase; I, II, III, IV and V: complexes I through V of the electron transport chain; Cit c: Cytochrome c; Cox5b: Cytochrome c oxidase subunit 5B; Cox7a2: Cytochrome c oxidase subunit 7A2; Atp5b: ATP synthase subunit beta; Atp5a1: ATP synthase subunit alpha; Gss: Glutathione synthetase; Gsr: Glutathione reductase.


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Figure 1



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Table 1: Body weight, food intake, fat depots weight, serum leptin and HOMA-IR of control and restricted groups Group

Control

Restricted

6.25 ± 0.07 (12) 5.15 ± 0.18*** (12) Body weight at birth (g) 86.2 ± 4.8 (5) 89.7 ± 0.8 (5) Body weight at weaning (g) 425.55 ± 13.21 (12) 422.46 ± 15.36 (12) Body weight at 4 months (g) 6.52 ± 0.23 (9) 6.05 ± 0.10 (9) Food intake at 4 months (g/100g) 1.06 ± 0.08 (12) 1.37 ± 0.08** (12) Retroperitoneal fat (g/100g) 1.21 ± 0.11 (12) 1.61 ± 0.07** (12) Gonadal fat (g/100g) 0.88 ± 0.07 (12) 0.93 ± 0.03 (12) Mesenteric fat (g/100g) 3.15 ± 0.18 (12) 3.91 ± 0.11** (12) Fat depots sum (g/100g) 1.14 ± 0.42 (9) 3.37 ± 0.93* (9) Leptin (ng/mL) 3.92 ± 0.52 (8) 6.62 ± 1.18 (9) HOMA-IR Values are means ± SEM; (number of animals). P-value for Student t test. * p < 0.05; ** p < 0.01; *** p < 0.001 versus the control group.


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Table 2: Significantly altered proteins in the hypothalamus of adult male rats submitted to intrauterine growth restriction compared to controls

UniProt ID

Gene name

B0BN85 M0RC99 P04218 P09951-2 P11951 P15650 P19643 P27881 P35171 P49242 P60881-2 P62744 P68403 Q62625-2 Q9JLJ3 Q9QZR6-4 Q9Z2F5 P01186 P62828 P11232 P10111 P60881

Sugt1 Rab5a Cd200 Syn1 Cox6c2 Acadl Maob Hk2 Cox7a2 Rps3a Snap25 Ap2s1 Prkcb Map1lc3b Aldh9a1 #Sept9 Ctbp1 Avp Ran Txn Ppia Snap25

Protein description Down-regulated Protein SGT1 homolog Ras-related protein Rab-5A OX-2 membrane glycoprotein Synapsin-1 (Isoform IB) Cytochrome c oxidase subunit 6C-2 Long-chain specific acyl-CoA dehydrogenase, mitochondrial Amine oxidase [flavin-containing] B Hexokinase-2 Cytochrome c oxidase subunit 7A2, mitochondrial 40S ribosomal protein S3a Synaptosomal-associated protein 25 (Isoform 2) AP-2 complex subunit sigma Protein kinase C beta type Microtubule-associated proteins 1A/1B light chain 3B (Isoform 2) 4-trimethylaminobutyraldehyde dehydrogenase Septin-9 (Isoform 4) C-terminal-binding protein 1 Vasopressin-neurophysin 2-copeptin GTP-binding nuclear protein Ran Thioredoxin Peptidyl-prolyl cis-trans isomerase A Synaptosomal-associated protein 25

FC

P-value

Not detected in the restricted group1

0.60 0.61 0.63 0.67 0.69

0.0433 0.0113 0.0331 0.0186 0.0175 33

ACS Paragon Plus Environment


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Table 2: Continued

O35987 P12075 P10362 Q9QXU9 P30904 P47728 Q9EQX9 Q64119 P10719 P05065 P52555 P70580 Q6P6R2 Q00981

Nsfl1c Cox5b Scg2 Pcsk1n Mif Calb2 Ube2n Myl6 Atp5b Aldoa Erp29 Pgrmc1 Dld Uchl1

B0BNE5 O88506 P11505-6 P46413 P62483 P62994-2 P70619 Q4V7D2 Q5RK09

Esd Stk39 Atp2b1 Gss Kcnab2 Grb2 Gsr Rogdi Eif3g

NSFL1 cofactor p47 Cytochrome c oxidase subunit 5B, mitochondrial Secretogranin-2 ProSAAS Macrophage migration inhibitory factor Calretinin Ubiquitin-conjugating enzyme E2 N Myosin light polypeptide 6 ATP synthase subunit beta, mitochondrial Fructose-bisphosphate aldolase A Endoplasmic reticulum resident protein 29 Membrane-associated progesterone receptor component 1 Dihydrolipoyl dehydrogenase, mitochondrial Ubiquitin carboxyl-terminal hydrolase isozyme L1 Up-regulated S-formylglutathione hydrolase STE20/SPS1-related proline-alanine-rich protein kinase Plasma membrane calcium-transporting ATPase 1 (Isoform K) Glutathione synthetase Voltage-gated potassium channel subunit beta-2 Growth factor receptor-bound protein 2 (Isoform 2) Glutathione reductase Protein rogdi homolog Eukaryotic translation initiation factor 3 subunit G

0.71 0.72 0.73 0.75 0.78 0.79 0.79 0.79 0.82 0.82 0.83 0.83 0.84 0.86

0.0180 0.0358 0.0008 0.0038 0.0399 0.0448 0.0023 0.0445 0.0005 0.0406 0.0175 0.0236 0.0077 0.0337

Not detected in the control group2


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Table 2: Continued

Q5XID1 Ciapin1 Anamorsin Q6PEC0 Nudt2 Bis(5'-nucleosyl)-tetraphosphatase [asymmetrical] Not detected in the control group2 Q9JJK1 Gpm6b Neuronal membrane glycoprotein M6-b Q9Z2L9 Ndrg4 Protein NDRG4 Q9Z339 Gsto1 Glutathione S-transferase omega-1 1.64 0.0359 P51635 Akr1a1 Alcohol dehydrogenase [NADP(+)] 1.44 0.0120 P13233 Cnp 2',3'-cyclic-nucleotide 3'-phosphodiesterase 1.39 0.0017 Q5M7A7 Cnrip1 CB1 cannabinoid receptor-interacting protein 1 1.37 0.0141 Q66HA6 Arl8b ADP-ribosylation factor-like protein 8B 1.36 0.0350 Q5XI78 Ogdh alpha-ketoglutarate dehydrogenase, mitochondrial 1.26 0.0024 P62815 Atp6v1b2 V-type proton ATPase subunit B, brain isoform 1.17 0.0055 P15999 Atp5a1 ATP synthase subunit alpha, mitochondrial 1.14 0.0264 Abbreviation: UniProt, Universal Protein Resource; FC, fold change (Restricted/Control). P-value for Student t test. 1 proteins not detected in any of the 12 replicates of the restricted group and identified in at least 4 replicates of the control group, indicating down-regulation by IUGR 2 proteins not detected in any of the 12 replicates of the control group and identified in at least 4 replicates of the restricted group, indicating up-regulation by IUGR.



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Table 3: Effect of intrauterine growth restriction on hypothalamic protein expression, as analyzed by either shotgun proteomics or two-dimensional gel electrophoresis UniProt ID

Protein name

P05065

Fructose-bisphosphate aldolase A1

Q00981 P15999 P51635

Shotgun proteomics FC P-value 0.82

0.041

2D-Gel FC 0.92 0.92

Ubiquitin carboxyl-terminal hydrolase 0.86 0.034 0.86 isozyme L1 1.19 ATP synthase subunit alpha, 1.14 0.026 1 mitochondrial 1.30 Alcohol dehydrogenase [NADP+] 1.44 0.012 1.02 Abbreviation: FC, fold change (Restricted/Control). P-value for Student t test. 1 Identified in two different spots in the 2D-Gel.

P-value 0.646 0.536 0.084 0.601 0.044 0.881


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Table 4: Significantly altered metabolites in the hypothalamus of adult male rats submitted to intrauterine growth restriction compared to controls KEGG Entry

C18043 C00016 C00300 C00149 C00025 C00130 C00253 C00035 C00387 C02727 C00242 C00031 C00043 C00170

HMDB ID

Compound

HMDB00653 HMDB01248 HMDB00064 HMDB00156 HMDB00148

Decreased levels Cholesterol sulfate Flavin adenine dinucleotide (FAD) Creatine Malate Glutamate

Single reaction monitoring (SEM) Q1/Q3

FC

P-value

465.2/97.0 786.0/348.0 132.0/90.0 133.0/115.0 148.1/84.1

0.61 0.64 0.85 0.87 0.91

0.0093 0.0128 0.0011 0.0246 0.0394

Increased levels HMDB00175 Inosinic acid (IMP) 349.0/137.0 1.64 HMDB01488 Niacin 124.1/78.1 1.44 HMDB01201 Guanosine 5'-diphosphate (GDP) 442.0/159.0 1.36 HMDB00133 Guanosine 284.1/135.0 1.32 HMDB00206 N6-Acetyl-L-lysine 189.0/84.2 1.29 HMDB00132 Guanine 152.2/110.0 1.24 HMDB00122 Glucose 179.1/89.1 1.23 HMDB00290 UDP-N-acetylglucosamine 606.0/385.0 1.19 HMDB01173 S-methyl-5-thioadenosine 298.0/136.0 1.17 Abbreviation: KEGG, Kyoto Encyclopedia of Genes and Genomes; HMDB, Human Metabolome Database ; FC, fold change (Restricted/Control). P-value for Student t test.

0.0028 0.0029 0.0157 0.0024 0.0009 0.0151 0.0096 0.0242 0.0268



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Table 5: Significantly altered metabolites in the serum of adult male rats submitted to intrauterine growth restriction compared to controls KEGG Entry

C05635 C00328 C00127 C00295 C00049 C04256 C00025 C00149 C03196 C00022 C01159 C00245 C00062 C00437 C19837 C00352 C01551 C01179 C00031

HMDB ID

Compound

HMDB00763 HMDB00684 HMDB03337 HMDB00226 HMDB00191 HMDB01367 HMDB00148 HMDB00156 HMDB00694 HMDB00243 HMDB01294 HMDB00251 HMDB00517 HMDB03357

Decreased levels 5-Hydroxyindoleacetic acid Kynurenine Glutathione disulfide Orotate Aspartate N-acetyl-glucosamine-1-phosphate Glutamate Malate 2-Hydroxygluterate Pyruvate 2,3-Diphosphoglyceric acid Taurine L-Arginine N-acetyl-L-ornithine

Single reaction monitoring (SEM) Q1/Q3

FC

P-value

192.0/146.0 209.0/146.0 613.0/231.0 155.0/111.0 134.0/74.0 300.0/79.0 148.1/84.1 133.0/115.0 147.1/128.7 87.0/43.0 265.1/167.1 124.0/80.0 175.0/60.0 175.0/115.1

0.44 0.50 0.55 0.59 0.65 0.67 0.72 0.75 0.78 0.79 0.81 0.83 0.86 0.86

8.46E-03 0.0434 0.0219 0.0002 0.0007 0.0032 0.0006 0.0236 0.0244 0.0327 0.0332 0.0454 0.0223 0.0410

Increased levels HMDB03320 Indole-3-carboxylic acid 160.0/116.0 1.41 HMDB01254 D-glucosamine-6-phosphate 260.0/126.0 1.22 HMDB00462 Allantoin 157.1/114.0 1.18 HMDB00707 Hydroxyphenylpyruvate 179.1/107.0 1.17 HMDB00122 Glucose 179.1/89.1 1.12 Abbreviation: KEGG, Kyoto Encyclopedia of Genes and Genomes; HMDB, Human Metabolome Database ; FC, fold change (Restricted/Control). P-value for Student t test.

0.0363 0.0284 0.0397 0.0148 0.0252


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Table 6: Integrated pathway analysis of significantly altered hypothalamic proteins and metabolites of adult rats submitted to intrauterine growth restriction

Pathway

Glycolysis/Glyconeogenesis

Oxidative phosphorylation

Synaptic vesicle cycle

Glutathione metabolism FoxO signaling pathway

Protein Dihydrolipoyl dehydrogenase, mitochondrial (↓) Hexokinase-2 (↓) Fructose-bisphosphate aldolase A (↓) 4-trimethylaminobutyraldehyde dehydrogenase (↓) Alcohol dehydrogenase [NADP(+)] (↑) Cytochrome c oxidase subunit 5B, mitochondrial (↓) Cytochrome c oxidase subunit 6C-2 (↓) Cytochrome c oxidase subunit 7A2, mitochondrial (↓) ATP synthase subunit alpha, mitochondrial (↑) ATP synthase subunit beta, mitochondrial (↓) V-type proton ATPase subunit B, brain isoform (↑) Synaptosomal-associated protein 25 (↓) V-type proton ATPase subunit B, brain isoform (↑) AP-2 complex subunit sigma (↓) Glutathione reductase (↑) Glutathione synthetase (↑) Glutathione S-transferase omega-1 (↑) Growth factor receptor-bound protein 2 (↑)

Metabolite

p-value q-value

Glucose (↑)

0.00001 0.00053

0.00011 0.00295

Glutamate (↓)

0.0010

0.0152

Glutamate (↓)

0.0012

0.0157

Glucose (↑) Glutamate (↓)

0.0014

0.0172



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Table 6: Continued

Purine metabolism

Bis(5'-nucleosyl)-tetraphosphatase [asymmetrical] (↑)

IMP (↑) Guanine (↑) Guanosine (↑) GDP (↑)

0.0038

Amine oxidase [flavin-containing] B (↓) alpha-ketoglutarate dehydrogenase, mitochondrial (↑) 0.0038 Tryptophan metabolism 4-trimethylaminobutyraldehyde dehydrogenase (↓) Dihydrolipoyl dehydrogenase, mitochondrial (↓) Malate (↓) 0.0047 Citrate cycle (TCA cycle) alpha-ketoglutarate dehydrogenase, mitochondrial (↑) Hexokinase-2 (↓) Fructose-bisphosphate aldolase A (↓) Glucose (↑) 0.0050 HIF-1 signaling pathway Protein kinase C beta type (↓) Amine oxidase [flavin-containing] B (↓) Creatine (↓) Arginine and proline 0.0053 metabolism 4-trimethylaminobutyraldehyde dehydrogenase (↓) Glutamate (↓) Amine oxidase [flavin-containing] B (↓) Glutamate (↓) 0.0054 Histidine metabolism 4-trimethylaminobutyraldehyde dehydrogenase (↓) Plasma membrane calcium-transporting ATPase 1 (↑) Endocrine and other factorProtein kinase C beta type (↓) 0.00574 regulated calcium reabsorption AP-2 complex subunit sigma (↓) Arrows indicates protein or metabolite down (↓) or up-regulation (↑). P-value for pathway over-representation analysis; q-value: corrected p-values (False Discovery Rate, Benjamini-Hochberg)

0.0328

0.0328 0.0365 0.038 0.0397 0.0399

0.0418


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Figure 1: Main effects of intrauterine growth restriction (IUGR) on hypothalamic proteome and metabolome of adult male rats. Proteins/metabolites in blue and red represent down and up-regulation, respectively. Hk2: hexokinase-2; Aldoa: Fructose-bisphosphate aldolase A; PDC: pyruvate dehydrogenase complex; Pdh: pyruvate dehydrogenase; Dlat:dihydrolipoamide acetyltransferase; Dld: Dihydrolipoyl dehydrogenase; AcCoA: acetyl-CoA; α-KG: alfa-ketoglutarate; AKDC = α-ketoglutarate dehydrogenase complex; Ogdh: alphaketoglutarate dehydrogenase; Dlst: dihydrolipoyl succinyltransferase; I, II, III, IV and V: complexes I through V of the electron transport chain; Cit c: Cytochrome c; Cox5b: Cytochrome c oxidase subunit 5B; Cox7a2: Cytochrome c oxidase subunit 7A2; Atp5b: ATP synthase subunit beta; Atp5a1: ATP synthase subunit alpha; Gss: Glutathione synthetase; Gsr: Glutathione reductase. 165x165mm (300 x 300 DPI)

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Integrated Analysis of Proteomic and Metabolomic D - ACS Publications

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