Quantitative proteomics reveals decreased expression of major urinary proteins in the liver of apoE/eNOS‐DKO mice
Summary: Endothelial nitric oxide synthase (eNOS)‐derived nitric oxide (NO) plays an important role, not only in endothelium‐dependent vasodilation but also in lipid and glucose homeostasis in the liver and exerts beneficial effects on mitochondrial biogenesis and respiration. Thus, the aim of our study was to use iTRAQ‐based quantitative proteomics to investigate the changes in protein expression in the mitochondrial and cytosolic fractions isolated from the liver of the double (apolipoprotein E (apoE) and eNOS) knockout (apoE/eNOS‐DKO) mice as compared to apoE KO mice (apoE−/−) – an animal model of atherosclerosis and hepatic steatosis. Collectively, the deficiency of eNOS resulted in increased expression of proteins related to gluconeogenesis, fatty acids and cholesterol biosynthesis as well as the decreased expression of proteins participated in triglyceride breakdown, cholesterol transport, protein transcription & translation and processing in endoplasmic reticulum (ER). Moreover, one of the most downregulated proteins were major urinary proteins (MUPs), which are abundantly expressed in the liver and were shown to be involved in the regulation of lipid and glucose metabolism. The exact functional consequences of the revealed alterations require further investigation.
apolipoprotein E; endothelial NOS; liver; major urinary proteins; proteomics
INTRODUCTION
Nitric oxide (NO) is a gaseous signaling molecule engaged in a variety of physiological and pathological processes.[1] It is endogenously biosynthesized from L‐arginine, oxygen and NADPH by nitric oxide synthase (NOS) enzymes. Three major isoforms of NOS are known – 2 constitutive: endothelial NOS (eNOS), and neuronal NOS (nNOS); and inducible NOS (iNOS).[2] It is recognized that eNOS‐derived NO not only plays multiple roles in proper functioning of cardiovascular system but also seems to be an important regulator of lipid and glucose metabolism in the liver: it increases glycolysis, lipolysis and fatty acid oxidation, as well as decreases triglyceride and de novo fatty acid synthesis, and inhibits gluconeogenesis.[3] Importantly, on the subcellular level NO has been shown to exert a profound effect on mitochondria: it reduces mitochondrial oxygen consumption and the formation of reactive oxygen species (ROS).[4] Noteworthy, hepatocyte mitochondrial dysfunction characterized by the augmentation of ROS production, impairment of fatty acid beta‐oxidation and enhancement of apoptosis has been recently proposed as a crucial factor in the development of nonalcoholic fatty liver disease (NAFLD). Indeed, eNOS‐deficient mice have been characterized by insulin resistance, hyperlipidaemia,[5] abnormal fat and glycogen deposition in the liver[6] and exacerbated early‐stage nonalcoholic fatty liver disease (NAFLD).[7] Furthermore, it has been observed that eNOS dysfunction is associated with NAFLD in humans.[8]
Apolipoprotein E (apoE) is a glycoprotein synthesized and secreted by hepatocytes, which is involved in the internalization and catabolism of lipoproteins in the liver. The apoE‐knockout mice (apoE−/−) spontaneously develop hypercholesterolaemia, dyslipidaemia, arterial lesions and represent a well known model of atherosclerosis, widely used in studies on its pathogenesis and pharmacotherapy.[9] [10] Moreover, they exhibit changes in the liver resembling hepatic steatosis and fibrosis.[11] To investigate the impact of eNOS deficiency on atherosclerosis development, the double (apoE and eNOS) knockout (apoE/eNOS‐DKO) mice were created. Interestingly, eNOS deficiency accelerated atherosclerosis and introduced peripheral coronary disease, chronic myocardial ischaemia, heart failure and other cardiovascular complications in apoE−/− mice.[12] However, so far the impact of eNOS deficiency on the steatotic changes in the liver was not explored in apoE−/− mice. Therefore, it would be interesting to investigate if the deficiency of eNOS in the liver leads to hepatic steatosis and alterations of proteins related to lipid and glucose homeostasis as well as mitochondrial function.
In this work we applied iTRAQ‐based quantitative proteomics to elucidate the changes in protein expression in the mitochondrial and cytosolic fractions isolated from the liver of apoE/eNOS‐DKO as compared to apoE−/− mice.
RESULTS
Histology results
Hematoxylin/eosin (HE) staining showed that lobular structure of the liver was preserved in both groups. Portal spaces were not enlarged, with no inflammatory infiltrates. In both groups less than 10% of hepatocytes had signs of granulation of the cytoplasm. The most striking difference between apoE−/− mice and apoE/eNOS‐DKO mice was focal presence of polymorphic, hyperchromatic and bi‐nucleated hepatocytes in the apoE/eNOS‐DKO group (Figure ).
Differential protein expression assessed by iTRAQ method
Protein expression in the liver of apoE−/− mice and apoE/eNOS‐DKO mice was evaluated using isobaric tag for relative quantitation (iTRAQ method). The quality of obtained data was visualized as histograms and multiple scatter plots, which represent the distribution of datasets (Figure A,B) and the robustness of measurements (Figure C,D), respectively. Collectively, 232 proteins in the cytosolic and microsomal fraction and 41 proteins in the mitochondria‐enriched fraction were differentially expressed in the liver of apoE/eNOS mice as compared to apoE−/− mice (Tables , ). The results were presented as Volcano plots based on log2 fold change and P‐value (Figure ) and heat maps, which show the most differentially expressed proteins in the cytosolic & microsomal fraction and mitochondria‐enriched fraction (Figure ). Importantly, iTRAQ‐based quantitative proteomic analysis revealed that different isoforms of major urinary proteins (MUPs): eg, MUP1 (UniProtKB accession number: P11588) and MUP3 (UniProtKB accession number: P04939) were the most downregulated proteins (2 times) in the liver of apoE/eNOS mice as compared to apoE−/− mice. These results were additionally confirmed by Western blot (Figure ). Furthermore, the most interesting differentially expressed proteins in the liver of apoE/eNOS‐DKO mice were listed in Table .
Selected, differentially expressed proteins in the liver of apoE/eNOS‐DKO mice with the indication of their biological function
| Biological process | Differentially expressed proteins |
|---|
| Fatty acids and triglycerides biosynthesis | Acetyl‐CoA synthetase ↑, NADP‐dependent malic enzyme ↑, fatty acid synthase ↑, acetyl‐CoA carboxylase 2/1 ↑, ATP‐citrate synthase ↑, glycerol kinase ↑ |
| Cholesterol biosynthesis | HMG‐CoA synthase ↑, SEC14‐like protein 2 ↑, isopentenyl‐diphosphate isomerase ↑, farnesyl pyrophosphate synthase ↑ |
| Triglycerides breakdown | Carboxylesterase 3A/B ↓, arylacetamide deacetylase ↓ |
| Cholesterol transport | Sterol carrier protein ↓, fatty acid‐binding protein ↓, apolipoprotein A‐I ↓, serum paraoxonase 1 ↓ |
| Gluconeogenesis & glucose homeostasis | Major urinary protein 1/3 ↓, glucokinase regulatory protein ↑, pyruvate carboxylase ↑, L‐serine dehydratase ↑, malate dehydrogenase (cytoplasmic) ↑, triosephosphate isomerase ↑, glycogen synthase ↓ |
| Protein synthesis and processing in endoplasmic reticulum and unfolded protein response (UPR) | Neutral alpha‐glucosidase AB ↓, calnexin ↓, hypoxia upregulated protein 1 ↓, protein disulfide‐isomerase ↓, dolichyl‐diphosphooligosaccharide‐protein glycosyltransferase ↓, protein ERGIC‐53 ↓, calumenin ↓, endoplasmin ↓, glucosidase 2 subunit beta ↓, vesicular integral‐membrane protein VIP36 ↓, 78 kDa glucose‐regulated protein ↓, peptidyl‐prolyl cis‐trans isomerase B ↓, ribosome‐binding protein 1 ↓, calreticulin ↓ |
| Protein Transcription & translation | 60S and 40S ribosomal protein ↓, elongation factor 1 (alpha, beta, gamma, delta) ↓, heterogeneous ribonucleoprotein particle ↓, TAR DNA – binding protein ↓, polypyrimidine‐track binding protein 1 ↓, eukaryotic translation initiation factor 3, subunit B ↓ |
| Response to oxidative stress | Catalase ↓, mitochondrial aldehyde dehydrogenase ↓, glutatione S‐transferase ↑, mitochondria peptide methionine sulfoxide reductase ↑, dimethylargininase 1 ↑ |
DISCUSSION
Nitric oxide (NO) is a crucial player not only in the cardiovascular system, but also in the liver, where it is involved in glucose and lipid metabolism.[3] [5] [6] [7] In the present study we used quantitative proteomics to elucidate the changes in the liver's protein expression in apoE/eNOS‐DKO mice as compared to apoE KO mice (apoE−/−) – an animal model of atherosclerosis and mild hepatic steatosis. The main finding of our work is that eNOS deficiency in the liver of apoE−/− mice led to the upregulation of proteins related to gluconeogenesis, fatty acids and cholesterol biosynthesis as well as the downregulation of proteins participated in triglyceride breakdown, cholesterol transport, protein transcription & translation and processing in endoplasmic reticulum (ER). Moreover, it seems plausible that some of those changes could be at least partially mediated by MUPs, which appear to be the most downregulated proteins in the liver of apoE/eNOS‐DKO mice (Figure ).
Major urinary proteins are small, highly similar lipocalin family members, expressed in hepatocytes and secreted into circulation.[13] They serve not only as pheromone carriers in rodents but also play a role in regulation of metabolism. It has been shown that MUP1 expression was significantly diminished in mouse models of genetic and dietary fat‐induced obesity and diabetes and its overexpression led to the reduction of glucose intolerance and hyperglycaemia.[14] [15] Furthermore, MUP1 inhibited the expression of key genes engaged in hepatic gluconeogenesis and lipogenesis in liver of db/db mice[15] and improved insulin sensitivity by increasing mitochondrial biogenesis and energy expenditure in skeletal muscles of these animals.[14] Noteworthy, we have previously observed enhanced expression of MUP2 in the liver of apoE−/− mice as compared to wild‐type mice, what might represent a compensatory mechanism to counteract the lack of apoE.[16] Here, we have shown that eNOS deficiency caused the 2‐fold decrease of MUP1 and MUP3 expression in the liver of apoE−/− mice, which was associated with the upregulation of proteins participated in hepatic gluconeogenesis and fatty acids and cholesterol biosynthesis. Whether MUPs are causally involved in gluconeogenic and lipogenic proteins expression in the liver of eNOS‐deficient apoE−/− mice this question remains open for further research.
As mentioned above, proteins engaged in hepatic gluconeogenesis, such as pyruvate carboxylase, L‐serine dehydratase, malate dehydrogenase (cytoplasmic) and triosephosphate isomerase were upregulated in the liver of apoE/eNOS‐DKO mice. It is well known that hepatic gluconeogenesis is greatly elevated in type 2 diabetes, which contributes to glucose intolerance and hyperglycaemia.[17] [18] Since NO has the capacity to regulate insulin resistance and obesity[19] its deficiency may constitute the pathological link leading to the development of diabetes and obesity. Consistently, we have also observed the increased expression of glucokinase regulatory protein (GKRP) and the decreased expression of glycogen synthase (GYS2) in the liver of apoE/eNOS‐DKO mice. GKRP is responsible for the regulation of hepatic glucose metabolism by controlling an activity and cellular localization of glucokinase – an enzyme that phosphorylates glucose, preparing it for glycogen synthesis or glycolysis.[20] It has been demonstrated that polymorphism or acetylation of GKRP were associated with type 2 diabetes.[21] [22] Similarly, the diminished expression of GYS2 – a protein engaged in glycogen biosynthesis – was observed in patients with type 2 diabetes.[23] Clearly, further investigations are required to elucidate the role of eNOS in the regulation of insulin resistance and its involvement in the development of diabetes.
Recently, it has been recognized that eNOS‐deficient mice exhibit insulin resistance, hyperlipidaemia,[5] abnormal fat and glycogen deposition in the liver[6] and exacerbated early‐stage nonalcoholic fatty liver disease (NAFLD).[7] Of note, our study point to the upregulation of proteins related to cholesterol biosynthesis (HMG‐CoA synthase, SEC14‐like protein 2, isopentenyl‐diphosphate isomerase, farnesyl pyrophosphate synthase) as well as fatty acids and triglycerides biosynthesis (acetyl‐CoA synthetase, NADP‐dependent malic enzyme, fatty acid synthase (FAS), acetyl‐CoA carboxylase 2/1 (ACC), ATP‐citrate synthase, glycerol kinase) and the downregulation of proteins related to triglycerides breakdown (carboxylesterase 3A/B (CES), arylacetamide deacetylase) and cholesterol transport (sterol carrier protein (SCP), fatty acid‐binding protein (L‐FABP), apolipoprotein A‐I (ApoA‐I), serum paraoxonase (PON1)) in the liver of apoE/eNOS‐DKO mice. These results are in line with other reports regarding the role of NO in the regulation of obesity.[19] [24] The elevated levels of HMG‐CoA synthase (cholesterol biosynthesis) and FAS (fatty acid biosynthesis) were demonstrated in obesity‐related type 2 diabetes[25] and hepatic steatosis.[26] Inhibition of ACC (fatty acid biosynthesis) led to the reduction of hepatic steatosis and the improvement of insulin sensitivity in rats.[27] Furthermore, mice deficient in ACC were protected from diabetes.[28] In turn, deficiency of carboxylesterases: CES1 and CES2, which metabolize triacylglycerols and cholesteryl esters, resulted in hepatic steatosis, obesity and hyperlipidaemia.[29] Noteworthy, the loss of proteins participated in cholesterol transport: SCP‐2 and L‐FABP induces lipid accumulation in hepatocytes[30] and higher levels of L‐FABP were observed in patients with NAFLD.[31] Interestingly, proteins involved in reverse cholesterol transport from tissues to the liver, such as ApoA‐I and PON1 (components of high‐density lipoprotein [HDL]) were diminished in the liver of apoE/eNOS‐DKO mice. The low levels of HDL were shown to be associated with increased risk of cardiovascular events in atherosclerosis[32] and type 2 diabetes.[33] Similarly, serum PON1 levels were decreased in patients with insulin resistance.[34] It is tempting to speculate that the decreased expression of ApoA‐I and PON1 in the liver of apoE/eNOS‐DKO mice may contribute to accelerated atherosclerosis and peripheral coronary disease observed in these mice.
It has recently been shown, that eNOS plays a key role in hepatocyte proliferation in response to partial hepatectomy in mice.[35] Interestingly, our results point to impaired hepatocyte proliferation in apoE/eNOS‐DKO mice as we observed focal presence of polymorphic, hyperchromatic and bi‐nucleated hepatocytes in this group in comparison to apoE−/− mice. Additionally, iTRAQ‐based quantitative proteomics revealed diminished expression of proteins involved in transcription and translation (eg, 60S and 40S ribosomal protein, elongation factor 1, heterogeneous ribonucleoprotein particle) and synthesis and processing of proteins in ER (Table ). Among them were proteins responsible for the regulation of unfolded protein response (UPR) and ER stress, such as 78 kDa glucose‐regulated protein (GRP78), disulfide‐isomerase and ribosome‐binding protein 1. GRP78 is an important activator of UPR and ER stress and surprisingly its expression was shown to be induced by eNOS activation.[36] Whether eNOS is involved in the activation of UPR and ER stress requires further investigation.
In conclusion, using iTRAQ‐based quantitative proteomics we have revealed that eNOS deficiency in the liver of apoE−/− mice led to the upregulation of proteins related to gluconeogenesis, fatty acids and cholesterol biosynthesis as well as the downregulation of proteins participated in triglyceride breakdown, cholesterol transport, protein transcription & translation and processing in ER. Moreover, one of the most downregulated proteins were MUPs, which are abundantly expressed in the liver and are involved in the regulation of lipid and glucose metabolism. The functional consequences of the revealed alterations require further research.
METHODS
Animal experiments
All animal procedures were performed conform the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes and approved by the Jagiellonian University Ethical Committee on Animal Experiments (no. 73/2011). Ten female apoE and eNOS‐double knockout mice on B6.129P2 background were created from apoE‐knockout and eNOS‐knockout mice by Jackson Laboratory (Bar Harbor, ME, USA) (project number 21536_BHSM). The apoE and eNOS PCR genotyping was performed according to company protocols (http://jaxmice.jax.org/protocolsdb/) (Figure ). Thirteen female apoE‐single knockout mice on B6.129P2 background were purchased from Jackson Laboratory. Mice were maintained on a 12 hour dark/12 hour light cycle in air‐conditioned rooms (22.5 ± 0.5°C, 50 ± 5% humidity) with access to food and water ad libitum. At the age of 8 weeks mice were put on chow diet made by Ssniff (Soest, Germany) for 4 months. At the age of 6 months mice were killed and 1000 UI of fraxiparine (Sanofi‐Synthelabo, France) was injected into the peritoneum. Next, the liver was dissected.
Histology of the liver
The liver tissue samples were formalin fixed, embedded in paraffin and 2 μm paraffin sections were stained with the hematoxilin‐eosin method as previously described.[37]
iTRAQ‐based quantitative proteomics
Sample preparation
The liver was homogenized and then cytosolic and microsomal fraction and mitochondria‐enriched fraction were isolated at 4°C from the freshly‐harvested liver as previously described.[37] Cytosolic and microsomal fractions and mitochondria‐enriched pellets were resuspended in 0.5 mL of lysis buffer (7 mol/L urea, 2 mol/L thiourea, 4% CHAPS, 1% DTT, the mix of protease inhibitors; Sigma, St Louis, MO, USA), vortexed, incubated at 25°C for 30 minutes and then centrifuged at 12 000 g for 15 minutes. The protein concentrations were determined in the harvested supernatants with the Bradford method.[38] One hundred micrograms of calculated protein content of each sample was precipitated overnight with ice‐cold acetone (Sigma) (1:6 v:v). Samples were centrifuged at 12 000×g for 10 minutes at 4°C. Acetone was carefully removed and precipitates were air dried for 10 minutes. Subsequently, samples were dissolved, reduced and alkylated as recommended by iTRAQ protocol (ABSciex). Proteins were digested with trypsin (Promega, Germany) overnight, with 1:50 (w:w) ratio, at 37°C. Samples (n = 3 per group) were labelled with iTRAQ reagents as recommended by producer and ordered as follows: apoE−/−: 113, 114, 115 and apoE/eNOS‐DKO: 116, 119, 121. Labelled samples were combined, and dried in a vacuum concentrator (Eppendorf, Germany). Next, peptides were dissolved in 5% ACN, 0.1% TFA and purified with C18 MacroSpin Columns (Harvard Apparatus). Eluate was dried in vacuum concentrator, reconstituted in 5% ACN, 0.1% FA, and subjected to SCX fractionation. Samples were loaded onto previously conditioned SCX Macrospin columns (Harvard Apparatus), flow‐through fractions were collected, and peptides were eluted in 11 consecutive salt steps (all mmol/L: 5, 10, 20, 40, 60, 80, 100, 150, 200, 300, 500 ammonium acetate in 5% ACN, 0.1% FA). Each sample resulted in a total of 12 SCX fractions.
LC‐MS analysis
Samples were concentrated on a trap column (Acclaim PepMap100 RP C18 75 μm i.d. × 2 cm column; Thermo Scientific Dionex). Each fraction was then injected on‐line on PepMap100 RP C18 75 μm i.d. × 15 cm column (Thermo Scientific Dionex) and peptides were separated in 90 minutes 7%‐55% B phase linear gradient (A phase – 2% ACN and 0.1% formic acid; B phase ‐ 80% ACN and 0.1% formic acid) with a flow rate of 300 nL/min by UltiMate 3000 HPLC system (Thermo Scientific Dionex) and applied on‐line to a Velos Pro (Thermo Scientific, USA) dual‐pressure ion‐trap mass spectrometer. The main working nanoelectrospray ion source (Nanospray Flex, Thermo Scientific) parameters were as follows: ion spray voltage 1.7 kV and capillary temperature 250°C. Spectra were collected in full scan mode (400‐1500 Da, 50 ms AT), followed by 1 HCD (higher energy collisional dissociation) and 1 CID MS/MS of the 5 most intense ions from the preceding survey full scan under dynamic exclusion criteria. The RAW files were processed by the EasierMgf software, which combines the low mass range data from each HCD MS/MS spectrum with the corresponding CID data for the same precursor into single MS/MS spectra.[39] These hybrid HCD‐extended MS/MS CID spectra were analyzed by the X!Tandem (The GPM Organization) and Comet search algorithms and validated with Peptide Prophet and iProphet under Trans‐Proteomic Pipeline (TPP) suite of software (Institute for Systems Biology, Seattle, WA, USA). Search parameters were set as follows: taxonomy, mouse (UniProtKB/Swiss‐Prot); enzyme, trypsin; missed cleavage sites allowed, 2; fixed modification, Methylthio(C); variable modifications, Methionine oxidation(M); iTRAQ8plex(K), iTRAQ8plex(N‐term), iTRAQ8plex(Y); parent mass error,−1.5 to + 3.0 Da; and peptide fragment mass tolerance, 0.7 Da. Quantitative information of the peptides data was extracted from the HCD MS[2] data by Libra software under Trans‐Proteomic Pipeline (TPP). Peptide False Discovery Rate (FDR) was estimated by Mayu (TPP) and peptide identifications with FDR below 1% were considered as correct matches. DanteR software[40] was used for statistical analysis of iTRAQ‐labelled peptides. In brief, data was log2 transformed, normalized using linear regression and ANOVA was performed at peptide level using a linear model with minimum 2 and maximum 500 peptides. Finally, the Benjamini & Hochberg FDR correction was used to adjust P‐values. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE[41] partner repository with the dataset identifier PXD006642.
Western blot
Samples, containing equal amounts of total protein (as estimated with the Bradford method) were mixed with gel loading buffer (50 mmol/L Tris, 10% SDS, 10% glycerol, 10% 2‐mercaptoethanol, 2 mg/ml bromophenol blue) in a ratio 4:1 (v/v) and incubated at 95°C for 5 minutes. Samples (25 μg of protein) were separated on SDS‐polyacrylamide gels (7.5%‐15%) (Mini Protean II, Bio Rad, Hercules, CA, USA) using the Laemmli buffer system and proteins were semidry transferred to nitrocelulose membranes (Amersham Biosciences, USA). Membranes were blocked overnight at 4°C with 5% (w/v) non‐fat dried milk in TTBS and incubated 3 hours at room temperature with specific primary antibodies: 1:200 ANTI‐MUP (Santa Cruz, Dallas, TX, USA; raised against full length mouse MUP1; due to the sequence homology >98% highly cross‐reactive to all MUPs), 1:1000 ANTI‐β‐actin (Sigma) then for 1 hour with HRP‐conjugated secondary antibodies (Amersham Biosciences). Bands were developed with the use of ECL‐system reagents (Amersham Biosciences). Rainbow markers (Amersham Biosciences) were used for molecular weight determinations. Protein pattern images were taken using an ImageQuant Las 500 (GE Healthcare, UK). Data analysis was performed using Image Lite Studio software (LI‐COR, USA). Results are presented as a mean + SE of the mean (SEM). The equality of variance and the normality of the data were checked and then the t test was used for statistical analysis of the data. P < .05 was considered to be statistically significant.
ACKNOWLEDGEMENTS
This study was supported by the grant from the National Science Centre (NCN): 2012/05/B/NZ4/02743. Aneta Stachowicz acknowledges the financial support from the Foundation for Polish Science (FNP).
CONFLICT OF INTEREST
The authors declare that there is no duality of interest associated with this manuscript. RO, JJ and RK were responsible for the conception and design of the study. AS, MS, AW and KK were responsible for analyses of the samples. AS, MS, MB and RO were responsible for the interpretation of the data. AS drafted the article. All authors revised the paper critically for important intellectual content and gave final approval of the version to be published.
Supporting Information
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PHOTO (COLOR): Representative images of hematoxilin and eosin staining in livers of 6‐months old (A) apoE−/− mice and (B) apoE/eNOS‐DKO mice. Arrows indicate bi‐nucleated hepatocytes. Magnification 40×
PHOTO (COLOR): The visualization of proteomic datasets. Histograms show the distribution of data of (A) the cytosolic and microsomal fraction and (B) mitochondria‐enriched fraction isolated from the liver of apoE−/− and apoE/eNOS‐DKO mice. Multiple scatter plots display the robustness of measurements of (C) the cytosolic and microsomal fraction and (D) mitochondria‐enriched fraction in apoE−/− and apoE/eNOS‐DKO mice. K1, K2, K3 indicate replicates in apoE−/− group and D1, D2, D3 indicate replicates in apoE/eNOS‐DKO group
PHOTO (COLOR): The Volcano Plot of differential proteins expression in the (A) cytosolic & microsomal fraction and (B) mitochondria‐enriched fraction isolated from the liver of apoE/eNOS‐DKO mice. The graph shows the log2 fold change of protein expression between apoE/eNOS‐DKO group and apoE−/− group vs P‐value. The dashed line indicates threshold 0.05 for P‐value; n = 3 per group
PHOTO (COLOR): Heat map presentation of a hierarchical cluster of the most differentially expressed and significantly changed proteins (fold change >1.22 and <−1.22; P < .05) in the (A) cytosolic and microsomal fraction and (B) mitochondria‐enriched fraction isolated from the liver of apoE/eNOS‐DKO mice as compared to apoE−/− mice. The green and red colours represent low and high expression levels, respectively
PHOTO (COLOR): Western blot of MUPs, HMGCS1 and β‐actin in the cytosolic and microsomal fraction of the liver of apoE−/− mice and apoE/eNOS‐DKO mice. Mean ± SEM; P < .05; n = 3 per group
PHOTO (COLOR): The scheme presenting the key aspects of the lack of eNOS in the liver of apoE−/− mice as revealed by our proteomic approach
PHOTO (COLOR): PCR genotyping of apoE/eNOS‐DKO mice. The left panel shows PCR products characteristic for apoE−/− (245 bp) and wild‐type (155 bp) mice. The right panel shows PCR products for eNOS−/− (258 bp) and wild‐type (337 bp) mice
By Aneta Stachowicz; Rafał Olszanecki; Maciej Suski; Anna Wiśniewska; Katarzyna Kuś; Magdalena Białas; Jacek Jawień and Ryszard Korbut
