Defining the Adipose Tissue Proteome of Dairy Cows to Reveal

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Article

Defining the Adipose Tissue Proteome of Dairy Cows to Reveal Biomarkers Related to Peripartum Insulin Resistance and Metabolic Status Maya Zachut J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.5b00190 • Publication Date (Web): 10 Jun 2015 Downloaded from http://pubs.acs.org on June 11, 2015

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Journal of Proteome Research

Defining the Adipose Tissue Proteome of Dairy Cows to Reveal Biomarkers Related to Peripartum Insulin Resistance and Metabolic Status

Maya Zachut1,* 1

Department of Ruminant Science, Institute of Animal Sciences, Volcani Center, P.O. Box 6,

Bet Dagan 50250, Israel. *Corresponding author: M. Zachut, Department of Ruminant Science, Institute of Animal Sciences, Volcani Center, P.O. Box 6, Bet Dagan 50250, Israel. Telephone: 972-89484434, e-mail: [email protected]

ABSTRACT: Adipose tissue is a central regulator of metabolism in dairy cows. However, little is known about the association between various proteins in adipose tissue and the metabolic status of peripartum cows. Therefore, the objectives were to: 1) examine total protein expression in adipose tissue of dairy cows, and 2) identify biomarkers in adipose that are linked to insulin resistance and to cows' metabolic status. Adipose tissue biopsies were obtained from 8 multiparous cows at -17 and +4 d relative to parturition. Proteins were analyzed by intensity-based, label-free, quantitative shotgun proteomics (nanoLC–MS/MS). Cows were divided into groups with insulin-resistant (IR) and insulin-sensitive (IS) adipose according to protein kinase B phosphorylation following insulin stimulation. Cows with IR adipose lost more body weight postpartum compared to IS cows. Differential expression of 143 out of 586 proteins was detected in prepartum vs. postpartum adipose. Comparing IR to IS adipose revealed differential expression of 18.9% of the proteins; those related to lipolysis (hormone-sensitive lipase, perilipin, monoglycerol lipase) were increased in IR adipose. In conclusion, we found novel biomarkers related to IR in adipose and to metabolic status that

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could be used to characterize high-yielding dairy cows that are better adapted to peripartum metabolic stress.

KEYWORDS: adipose, proteome, dairy cow, insulin resistance

INTRODUCTION

Adipose tissue has two major functions: it regulates energy storage by storing and releasing fatty acids, and it serves as a major endocrine organ with a profound influence on metabolism and body homeostasis by secreting and regulating numerous molecules, hormones and adipokines.1,2 Many adipokines that contribute to inflammation and oxidative stress activate intracellular pathways that promote the development of insulin resistance;3 however, the role of specific proteins in the mechanisms involved in adipose tissue dysfunction is not well defined.1 In the high-yielding dairy cow, adipose tissue has a major role in the metabolic adaptations to support milk production during the transition from late pregnancy to calving and onset of lactation. During that period, there is a shift from a nonlactating lipogenic state to a period of tremendous energy demand for milk production, which results in massive lipolysis of adipose tissue.4,5 In addition, the multiple adaptations from late pregnancy to lactation in dairy cows are at least partly mediated by the development of insulin resistance in maternal peripheral tissues.5 There is great variation in the responses of individual cows to metabolic stress during the transition to lactation, which will affect performance throughout the lactation. It is well known that cows in the same herd consuming the same diet demonstrate large variations in feed intake, milk yield, body weight (BW) gain and energy balance.6 Furthermore, the activity of hormone-sensitive lipase (HSL), the rate-limiting enzyme for triglyceride


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hydrolysis which is regulated by insulin,1 and the rate of lipolysis have been reported to be highly related to the cow's genetic merit and actual milk production.7 In our previous study, we reported that a subgroup of cows exhibit insulin resistance in adipose tissue during the peripartum period (termed insulin-resistant, IR) based on phosphorylation of protein kinase B (Akt) in response to insulin stimulation, and that these cows also lost more BW postpartum; another subgroup of cows showed intact insulin signaling in adipose tissue (termed insulinsensitive, IS), and lost less BW.8 These findings suggest that the cow-specific response in adipose tissue (IR or IS) is correlated to the animal's metabolic status. Taken together, we hypothesize that the variation in response to peripartum metabolic stress will be reflected in adipose tissue function; hence, various proteins in the adipose tissue could serve as biomarkers that are linked to the cow's metabolic status. Proteomic techniques provide a valuable tool for identifying new adipokines as well as novel molecular markers in adipose and other tissues.9 To the best of our knowledge, there are no data from proteomic analyses of adipose tissue from the peripartum dairy cow, and little is known about the role of specific proteins in IR adipose and the association with metabolic status. Therefore, the objectives of this study were to: 1) provide new data on total protein expression in adipose tissue of peripartum high-yielding dairy cows, and 2) identify biomarkers in adipose tissue that are linked to insulin resistance and to the cow's metabolic status.



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MATERIALS AND METHODS

Animals

The experimental protocol for the study was approved by the Volcani Center Animal Care Committee and was conducted at the Volcani Center experimental farm in Bet Dagan, Israel. Eight high-yielding, 261 ± 5 d pregnant nonlactating Israeli-Holstein dairy cows averaging 740 ± 73 kg of BW and average lactation number 4.6 (range 3 - 6), participated in this study. Cows were group-housed in loose covered pens with adjacent outside yards and fed ad libitum once a day at 1100 h with a standard Israeli diet. After calving, milk production and BW were recorded electronically thrice daily (SAE, Kibbutz Afikim, Israel). Blood samples for nonesterified fatty acid (NEFA) analysis were collected thrice weekly from 21 d before expected calving until 21 d postpartum from the jugular vein into vacuum tubes containing lithium heparin (Becton Dickinson Systems, Cowley, England). The blood samples were collected after the morning milking at 0800 h, and plasma was separated after centrifugation at 4000 × g for 15 min and then stored at -32°C pending analysis. In this study cows were divided into two subgroups according to phosphorylation of protein kinase B (Akt) in adipose tissues in response to insulin stimulation; as it was found that adipose tissues of one subgroup of cows was insulin resistant based on lack of Akt phosphorylation (IR, n = 4), whereas the adipose tissues of the other subgroup was insulin sensitive (IS, n = 4).8


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Adipose Tissue Biopsies and Sample Processing

Adipose tissue biopsies were taken from each cow at two time points: late pregnancy (261 ± 5 d of pregnancy, on average 17 d prepartum) and early (3–5 d) postpartum. Adipose tissue samples were taken from the subcutaneous fat pad around the pin bones as previously described,8 immediately frozen in liquid nitrogen and stored at -80°C. For protein extraction, ~40 mg adipose tissue was homogenized for 3 min with two metal beads (5 mm in diameter, Eldan Technologies, Petah Tikva, Israel) in 1 mL of prechilled lysis buffer containing protease and phosphatase inhibitors (Sigma Aldrich) as described.8 Samples were incubated for 1 h at 4oC under continuous shaking, and then the homogenate was centrifuged at 20,000 × g for 15 min at 4oC to remove lipids and other particulate matter. The liquid phase was collected and stored at -80oC.

Sample Preparation for Proteomic Analysis

Protein concentration in each sample was determined using the bicinchoninic acid (BCA) assay. Samples were then subjected to in-solution tryptic digestion. Protein was first reduced by incubation with 5 mM dithiothreitol (Sigma) for 30 min at 60°C, and then alkylated with 10 mM iodoacetamide (Sigma) in the dark for 30 min at 21°C. Proteins were then subjected to digestion with trypsin (Promega) at a 1:50 trypsin-to-protein ratio for 16 h at 37°C. Detergents were then cleared from the samples using commercial detergent-removal columns (Pierce), and were desalted using a solid-phase extraction column (Oasis HLB, Waters). Digestions were stopped by 1% trifluroacetic acid. The samples were stored at -80oC until further analysis.



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Liquid Chromatography

ULC–MS-grade solvents were used for all chromatographic steps. Each sample was loaded onto a split-less nano-ultra performance liquid chromatography (UPLC) (10 kpsi nanoAcquity, Waters). The mobile phases were: (A) H2O + 0.1% (v/v) formic acid and (B) acetonitrile + 0.1% formic acid. Samples were desalted online using a reverse-phase C18 trapping column (180-µm internal diameter, 20-mm length, 5-µm particle size; Waters). The peptides were then separated using a HSS T3 nano-column (75-µm internal diameter, 250mm length, 1.8-µm particle size; Waters) at 0.35 µL/min. Peptides were eluted from the column into the MS using the following gradient: 4% to 35% solution B in 150 min, 35% to 90% B in 5 min, maintained at 95% for 5 min, and then back to initial conditions.

Mass Spectrometry

The nano-UPLC was coupled online through a nano-ESI emitter (10-µm tip; New Objective, Woburn, MA, USA) to a quadrupole orbitrap mass spectrometer (Q Exactive Plus, Thermo Scientific) using a FlexIon nanospray apparatus (Proxeon). Data were acquired in data dependent acquisition (DDA) mode, using a Top20 method. Quadrupole isolation window was set to 1.5 mass units, MS1 resolution was set to 60,000 (at 400 m/z) and maximum injection time was set to 120 ms. MS2 resolution was set to 17,500 and a maximum injection time of 60 ms, normalized collision energy was set to 26. Singly charged ions were excluded and dynamic exclusion was set to 80 sec.


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Data Processing and Analysis

Raw data were processed as recently reported10. Briefly, raw data were imported into Expressionist® software (Genedata). Data were filtered, smoothed, and aligned in retention time. This was followed by feature detection based on peak volume in RT, m/z and intensity space as well as isotopic clustering. A master peak list was generhated from all MS/MS events and sent for database searching using Mascot v2.5 (Matrix Sciences). Data were searched against the Bos taurus sequences in UniprotKB (http://www.uniprot.org/), version 20014_07, appended with 125 common laboratory-contaminating proteins for a total of 17,955 entries. Fixed modification was set to carbamidomethylation of cysteines and variable modification was set to oxidation of methionines. Search results were then imported back to Expressions to annotate the identified peaks. Proteins were then grouped based on shared peptides and identifications were filtered such that the global false-discovery rate was 0.6%. Data were normalized based on the total ion current. Protein abundance was obtained by the iBAQ method (sum of all peptide intensities per protein divided by the theoretical number of tryptic peptides for the particular protein). Principal component analysis (PCA) was used to assess global integrity of the data and search for outlier samples.

Bioinformatics Analysis

Differentially expressed proteins (DEPs) were analyzed through the use of Qiagen’s Ingenuity® Pathway Analysis (IPA®, Qiagen Redwood City, www.qiagen.com/ingenuity), to determine the most significantly relevant networks, pathways and biological functions.



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Western-Blot Analysis for Validation

For western blotting, protein concentration of the sample homogenate was measured according to Bradford (Bio-Rad protein quantification kit). Then, 20 µg of sample in Laemmli loading buffer was resolved by SDS-PAGE under reducing conditions, and transferred onto a nitrocellulose membrane with the following antibodies: monoacylglycerol lipase (1:1000, ab24701, Abcam Biotech Co., Cambridge, UK), HSL (1:1000, #4107, Cell Signaling Technology Inc.), perilipin (1:2000, #9349, Cell Signaling Technology Inc.), and actin (1:1000, ab46805, Abcam Biotech Co.). Enhanced chemiluminescence reaction was used for protein detection. Data were processed and analyzed by densitometry using ImageJ software (NIH, Bethesda, MD, USA). To ensure that quantitative data were obtained, chemiluminescence signals were measured under at least five consecutive exposure times to determine the linear range of signal intensity of each antibody. Specific band signals were normalized to actin as an internal standard.

Quantitative Real-Time PCR for Validation

For RNA extraction, ~40 mg of adipose tissue samples were homogenized with 1 metal bead in 1 ml of lysis solution according to the RNeasy lipid tissue mini kit (Qiagen GmbH, Hilden, Germany). First-strand cDNA was generated by a cDNA reverse transcription kit (Applied Biosystems Inc., Foster City, CA). Quantitative detection of specific mRNA transcripts was carried out by real-time PCR using a StepOnePlus instrument (Applied Biosystems Inc.) using SYBR green PCR mix (Invitrogen Corp., Carlsbad, CA). Primers (400 nM) of HSL, PLIN and the adipose tissue house-keeping gene bovine ribosomal S2 unit (BRPS2) were prepared according to Rocco and McNamara (2013),11 and the primer for MGLL was


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prepared according to Khan et al. (2013).12 Primers were validated before use, and data were normalized for the content of BRPS2 mRNA in adipose samples.

Statistical Analysis

Proteomics data, after logarithmic transformation, were analyzed by two-way ANOVA (Matlab software, 8.0.0.783) to measure the effects of time (prepartum vs. postpartum), subgroup (IR vs. IS) and their interaction. DEPs for each effect were determined by P-value < 0.05 and an absolute fold change (FC) > 1.5. BW differences in the present experiment and protein abundance from western blots and relative quantity of mRNA were analyzed with the GLM of SAS (version 9.2, 2002). BW loss across lactations and plasma NEFAs were analyzed as repeated measurements with the MIXED procedure, version 9.2 (SAS, 2002).

RESULTS

Protein Expression in Adipose Tissue of Peripartum Dairy Cows

We examined the expression of proteins in adipose tissue obtained from 8 high-yielding cows 17 d prepartum (261 d pregnant) and 4 d postpartum (in total 16 samples). After proteomic analysis, PCA showed that one sample (cow number 4, prepartum) was significantly different from all of the other samples and therefore was excluded from further analysis. In total, 15 samples (7 prepartum and 8 postpartum) were subjected to statistical analysis. A total of 586 proteins were identified and quantified in adipose tissues. Among them, 455 proteins were classified by Ingenuity® into main protein categories based on their function and into cellular



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locations, including the cytoplasm (n = 235), extracellular space (n = 94), nucleus (n = 62), plasma membrane (n = 56), and unknown location (n = 8) according to the Ingenuity Knowledge Base using the IPA® Software Package (Table 1). The function and/or location of the remaining proteins in our study could not be annotated, and therefore only these 455 proteins were subjected to downstream analysis.

Protein Expression Differences Between Prepartum and Postpartum Adipose Tissue

As adipose tissue plays a main role in the metabolic adaptations from late pregnancy to onset of lactation, we first compared the expression of proteins in prepartum vs. postpartum adipose samples. Out of 586 proteins, 143 (24.4%) were differentially expressed in adipose tissues prepartum compared to postpartum (P < 0.05 and FC > ± 1.5). The list of these proteins appears in Supplementary Table 1. Functional analysis of the DEPs was performed according to time period (pre- or postpartum). In total, 106 DEPs were subjected to IPA®. The most relevant molecular and cellular pathways that were enhanced postpartum compared to prepartum are shown in Figure 1. Among them were signaling and remodeling of epithelial adherens junctions, actin cytoskeleton signaling, and tight-junction signaling. Functional analysis (Ingenuity®) revealed 18 lipid metabolism-related functions that were significantly changed postpartum compared to prepartum, such as fatty-acid metabolism, esterification of lipids and oxidation of fatty acids. In accordance with these lipid-related functions, the expressions of several proteins related to lipid metabolism were decreased postpartum as compared to prepartum (Supplementary Table 1), among them fatty acid synthase (FAS, FC = - 4.7, P < 0.006), complement C3 (FC = - 3.0, P < 0.02) and acyl-CoA desaturase (SCD, FC = - 3.1, P < 0.02). The expression of annexin-A1 (ANXA1, FC = - 3.1, P < 0.02) was also lower in postpartum vs. prepartum adipose.


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Increased Lypolitic Rate Postpartum in Cows with IR Adipose

Daily BW data was analyzed from all cows postpartum. As shown in Figure 2A, although the average BW at week 1 postpartum was similar in IR and IS cows, those with IR adipose lost more BW postpartum compared to cows with IS adipose. In fact, by week 4 postpartum cows with IR adipose had lost 11.7% of their BW at calving, whereas cows with IS adipose lost only 5.5% of their BW (P < 0.1). The higher BW loss in cows with IR adipose inferred increased lipolysis, which was further supported by numerically (28%) increased plasma NEFA concentrations during the period of 3 weeks prepartum to 4 week postpartum in cows with IR adipose (Figure 2B, P < 0.2). One of the basic premises of the present study was that adipose tissue function, as partly reflected by BW loss postpartum, has a genetic basis in dairy cows; hence, we calculated BW loss during the first month postpartum of the cows that participated in this study (n = 8) during all of their lactations (from first to fifth lactation). As shown in Figure 3, in all lactations (1–5), the cows with IR adipose lost more BW than those with IS adipose during the first month postpartum (60.0 vs. 37.3 kg, respectively, SEM = 5.5, P < 0.027). This is in agreement with our hypothesis that adipose tissue function, as expressed by lipolysis rate during the transition period, could be an intrinsic trait characterizing the cow that is repeated throughout its life span.

Protein Expression Differences Between IR and IS Adipose Tissue

We examined the differential expression of adipose proteins in IR vs. IS (pre- and postpartum, i.e. effect of subgroup) and found that 111 out of 586 proteins, representing 18.9% of the total proteins detected in the adipose tissue, were differentially expressed (P < 0.05 and FC > ± 1.5, Figure 4 and Supplementary Table 1). Of these DEPs, the expression of 106 of them was



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increased in IR vs. IS adipose, whereas only 5 DEPs had decreased expression in IR compared to IS adipose. Functional analysis (Ingenuity®) revealed that the most relevant pathways according to the DEPs in IR vs. IS adipose were gluconeogenesis and glycolysis, 14–3–3-mediated signaling, TCA cycle and ERK/MAPK signaling (Figure 5). The most relevant functions of the DEPs in IR vs. IS adipose according to IPA® were inflammatory response and organization of actin cytoskeleton, whereas the most relevant lipid-related functions were accumulation of lipid, release of lipid, and lipolysis of adipose tissue (Table 2). One of the most relevant networks according to IPA® was related to lipid metabolism, and radial display of this network demonstrated that Akt is the main contact between the DEPs in this network (Supplementary Figure 1); this was of special interest since adipose was classified based on Akt phosphorylation in response to insulin stimulation in this tissue.8

Biomarkers in Adipose that Are Linked to IR and Metabolic Status

The abundance of a number of proteins related to lipid metabolism was higher in IR compared to IS adipose: monoglyceride lipase (MGLL, FC = 8.2, P < 0.0003), perilipin (PLIN, FC = 1.5, P < 0.05) and FAS (FC = 6.1, P < 0.03). The increased expression of MGLL in IR adipose was also validated by western blot (P < 0.02, Figure 6A). The expression of HSL was higher in IR adipose (FC = 6.8, P < 0.03); however, the time-bysubgroup interaction was significant (P < 0.04). We therefore tested HSL expression at each time point by a separate t-test. The expression of HSL prepartum tended (P < 0.07) to be higher in IR vs. IS adipose, whereas postpartum its expression in IR and IS adipose was similar (P = 0.5). This pattern of expression was validated by western blots of HSL (Figure 6B). The differential expression of perilipin in IR and IS adipose was also validated by


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immuno-blots; however this was not significant (P < 0.15) probably due to the difficulty to detect differences in protein abundance by western blots in this fold change (1.5). In order to further validate the expression of these proteins in adipose tissues, we also examined the relative mRNA quantity of HSL, perilipin and MGLL in samples. Indeed, these genes were expressed in all samples and in addition the relative quantities (RQ) of HSL, perilipin and MGLL mRNA in adipose were numerically but not significantly higher in IR compared to IS cows: for HSL - RQ = 1.91 in IR vs. 1.24 in IS (SE = 0.52, P < 0.4); for perilipin - RQ = 1.61 in IR vs. 0.85 in IS (SE = 0.39, P < 0.5); and for MGLL - RQ = 1.00 in IR vs. 0.85 in IS (SE = 0.30, P < 0.7). Other interesting proteins that had increased expression in IR vs. IS adipose were tubulin (TUBB, FC = 3.4, P < 0.006), tubulin beta-1 chain (TUBB1, FC = 3.0, P < 0.05), tubulin beta-6 chain (TUBB6, FC = 3.5, P < 0.03), tubulin beta-2B chain (TUBB2B, FC = 2.9, P < 0.02), 14–3–3 protein gamma (WHAG, FC = 3.8, P < 0.0006), and ANXA1 (FC = 2.5, P < 0.03). The expression of clathrin heavy chain 1 (CLTC, FC = 3.8, P < 0.06) tended to be higher in IR vs. IS adipose.

DISCUSSION

The Unique Proteome of Adipose Tissue in Dairy Cows Compared to Beef Cattle

Knowledge of the proteome of adipose tissue, a central endocrine organ that has an important role in energy partition in dairy cows, especially during the periparturient period, is highly valuable for improving our understanding of the complex processes that govern overall metabolism in high-yielding dairy cows. This study revealed 586 proteins, 455 of which were classified into main protein categories based on their function and cellular localization by



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Ingenuity®. The proteome of bovine adipose tissue has been recently described in beef cattle;13 these animals are very different both genetically and physiologically from our modern high-yielding dairy cows, which produce up to 11,500 kg milk per lactation. As mentioned, the adipose tissue in high-yielding dairy cows has a major role in the homeorhetic adaptations to support milk production during the transition from late pregnancy to early lactation,5 and this is not the case in beef cattle adipose. The distinctive protein expression pattern in the adipose of dairy cows was demonstrated by comparing the overall protein expression in our study with that listed for beef adipose; only 148 proteins were shared between beef adipose (from a total of 637 classified proteins)13 and our dairy cow adipose data (455 classified proteins), which is only 23–32% of total classified proteins in adipose tissue. This difference could be partly attributed to differences between the methods of protein detection; nevertheless, the strong implication is that the proteome of adipose in high-yielding dairy cows is indeed unique. Although the list of proteins was distinct from that of beef adipose, the percentages of proteins within each function and cellular location (Table 1) were comparable in the two tissues;13 for example, we found that 51.6% of the proteins were located in the cytoplasm compared to 57.3% in beef adipose, 12.3% of proteins were located in the plasma membrane compared to 11.9% in beef, and 13.6% of the proteins were located in the nucleus compared to 8.9% in beef. In addition, the distinctive functions within each location (Table 1) were comparable to those found in beef adipose;13 for instance, within the plasma membrane, we identified 16.07% of proteins as enzymes compared to 18.42% in beef adipose, and we identified 5.36% of proteins as transmembrane receptors compared to 7.89% in beef adipose. These comparable findings suggest that our data indeed portrays the overall protein expression in dairy cow adipose, and the dissimilarities with other bovine species could be


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related to the actual function of the proteins within the tissue and an association with the difference in production designation—milk vs. meat.

Protein Expression Differences Between Prepartum and Postpartum Adipose Tissue

The abundance of one-fourth (24.4%) of the total proteins differed between prepartum and postpartum adipose. This magnitude of change reflects the major importance of the adipose tissue in the dynamic metabolic adaptations to lactation. In addition, some of the most relevant molecular and cellular pathways that were enhanced postpartum compared to prepartum were signaling and remodeling of epithelial adherens junctions, actin cytoskeleton signaling, and tight-junction signaling, which could all be related to remodeling of the adipose tissue during the massive lipolysis that occurs postpartum. The adipose tissue of dairy cows during late pregnancy (260 d of pregnancy) is in a state of lipogenesis, storing energy in the form of triacylglycerols as cows are generally in a state of positive energy balance; during early lactation, the adipose tissue undergoes massive lipolysis to support milk production. Indeed, the abundance of FAS, complement C3 and SCD, which are related to lipid metabolism in adipocytes, was lower in adipose tissue of postpartum vs. prepartum cows (Supplementary Table 1). Our data demonstrate the vast shift in protein expression during the transition from late pregnancy to early lactation, which is part of the homeorhetic adaptations undergone by the adipose tissue to sustain high-milk production in dairy cows. Another interesting finding is that the abundance of ANXA1 was higher in prepartum compared to postpartum adipose. Proteome analysis of beef cattle adipose tissue showed increased expression of ANXA1 in the adipose tissue of steers with greater back-fat thickness, suggesting ANXA1 as a protein marker for assessing animals with different back-fat thicknesses.14 Our results further support the positive correlation between adiposity and



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ANXA1 expression in adipose tissue, since cows during late pregnancy have more subcutaneous adipose tissue than postpartum cows; thus, ANXA1 could be a biomarker of adiposity in bovine species.

Protein Expression in IR vs. IS Adipose Tissue

We found that about one-fifth (18.9%) of the proteins expressed in the adipose tissue are differentially expressed in IR vs. IS adipose. This indicates that many proteins participate in the alterations in adipose function related to insulin resistance in periparturient dairy cows. In addition, the expressions of most of the DEPs (96.3%) increased, and others did not consistently decrease, in IR compared to IS adipose. This is in agreement with other studies reporting that proteins involved in lipid metabolism are mostly increased in adipose tissue in IR states.11–14 The specific role of several novel proteins that were differentially expressed in the IR adipose of dairy cows is discussed below.

Elevated Expression of Lipid Metabolism-Related Proteins in IR Adipose Tissues of Dairy Cows

The expression of a number of proteins related to lipid metabolism—i.e., MGLL, PLIN, FAS and HSL—was higher in IR compared to IS adipose in the present study. As insulin is an anabolic hormone, disruption of its intercellular signaling in IR adipose tissue will encourage increased lipolysis.1 Indeed, we have shown that cows with IR adipose lose more BW during early lactation (Figure 2A), which is in agreement with the increased lipolysis and higher expression of MGLL, PLIN and HSL in IR adipose in the present study. MGLL participates in lipolysis through mono-acylglycerol hydrolysis in adipose tissue,19 and in humans, MGLL


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is differentially expressed in obese compared to nonobese individuals,20 which fits the model of increased lipolysis in IR adipose in dairy cows. We also found increased expression of PLIN in IR adipose, which is in accordance with another study in which the expression of PLIN was higher in human IR subcutaneous adipose tissue compared to lean IS subjects.15 PLIN has a complex role in regulating both basal and stimulated adipocyte lipolysis;19 under unstimulated conditions, the presence of PLIN coating the lipid droplet functions as a protective barrier that restricts access of triacylglycerol lipases to neutral lipid substrates to prevent unrestrained basal lipolysis.21 However, under stimulated adipocyte lipolysis, PLIN has a different role, as protein kinase A (PKA)-dependent phosphorylation of PLIN may facilitate interaction with HSL on the lipid droplet, thereby increasing the activity of the enzyme.22 Taken together, PLIN expression and phosphorylation state are critical regulators of lipolysis in adipocytes;19 therefore, the higher expression of PLIN in IR adipose could be linked to the increased lipolysis observed in these cows. In this study, the expression of FAS was increased in IR compared to IS adipose. In contrast, a study in mice reported decreased expression of FAS in IR adipose.18 As FAS is a lipogenic enzyme that supports storage of fatty acids within the adipocyte, it is not clear why its expression was elevated in IR adipose in the current study. The abundance of HSL was higher in IR compared to IS adipose; however, a significant subgroup-by-time interaction indicated a different HSL-expression pattern at each time point. Prepartum HSL tended to be higher in IR adipose, whereas 4 d postpartum, its abundance was similar in IR and IS; these findings imply that the adipose tissue was in a state of lipolysis at 260 d of pregnancy in cows with IR adipose. On the other hand, the similar expression of HSL postpartum can be explained by the increased lipolysis that occurs in all cows in early lactation as shown in Figure 2B. Hence, our data suggest that HSL is a biomarker of IR only in prepartum adipose, and perhaps can be used as a biomarker of IR in



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later stages of lactation, such as wk 4 postpartum, when the differences between BW loss in the subgroups re-emerge (Figure 2A). HSL-mediated lipolysis is important to adipose fatty acid liberation in adipocytes.19 Catecholamines are the primary activators of fasting-induced lipolysis, through binding to beta-adrenergic receptors on the plasma membrane of adipocytes, which induces an increase in cAMP that activates PKA.19 PKA catalyzes the polyphosphorylation of HSL at multiple sites; this activates HSL and subsequent translocation to the lipid droplet, along with phosphorylation of PLIN, which together enhance lipolysis.19 The activity of HSL and the rate of lipolysis are highly related to genetic merit and actual milk production in the cow.7 Moreover, even though HSL catalyzes lipolysis, only about 12– 17% of the variation in stimulated lipolysis can be explained by an increase in HSL mRNA. It was therefore suggested that most of the control of HSL activity is post-translational.23 The results of the present study further support the role of HSL protein on overall lipolysis in dairy cows.7,23 Taken together, our proteomic analysis revealed several novel biomarker proteins related to lipid metabolism (MGLL, FAS, PLIN, HSL), which can serve as biomarkers of IR adipose and of metabolic status in dairy cows.

Other Possible Biomarkers of IR in Adipose Tissue of Dairy Cows

In the present study, the expressions of several proteins related to the glucose transporter GLUT4's translocation within the adipocyte increased (or tended to increase) in IR adipose: TUBB, WHAG, clathrin, actin, and three tubulin-family proteins (TUBB1, TUBB6, TUBB2B). In addition, the expression of fatty acid-binding protein 4 (FABP4, AP-2) was numerically higher in IR than in IS (FC = 2.1, P < 0.2). In adipocytes, insulin promotes glucose disposal primarily through GLUT4, a constitutively recycling membrane protein; defects in its cycling are associated with IR.24,25 GLUT4 internalization in adipocytes occurs


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partly by clathrin-mediated endocytosis (CME).24 Tubulins are a component of the microfilaments involved in intracellular trafficking. Several studies have reported longdistance linear movement of GLUT4 vesicles that coincides with the position of tubulin, and therefore microtubules could serve as tracks for the long-range trafficking of vesicles to and from the plasma membrane.25 Alternatively, microtubules could coordinate their function with the actin cytoskeleton, potentially handing on vesicles to cortical actin filaments.25 Based on this, we postulate that the increased expression of tubulins in IR adipose could be related to elevated GLUT4 endocytosis. The tendency for higher expression of clathrin (P < 0.06) and of structural proteins that are related to CME such as actin, tubulins and possibly AP-2, might also suggest that in IR adipose there is increased CME of GLUT4, as one of the mechanisms participating in the nonresponsiveness to insulin within the adipocyte. Moreover, IPA® revealed 5 DEPs in IR adipose related to 14–3–3 signaling: TUBB, TUBB1, WHAG, TUBB6, and TUBB2B. WHAG is a regulatory protein that binds many functionally diverse signaling proteins, including kinases, phosphatases, and transmembrane receptors; it is involved in insulin-regulated GLUT4 trafficking in adipocytes.26 The increased expression of WHAG in IR adipose in our study is in accordance with another study that reported a higher abundance of WHAG in subcutaneous and visceral adipose tissues of obese compared to nonobese subjects.20 Collectively, in this work, we identified several biomarkers that were elevated in IR adipose and are related to GLUT4 endocytosis. Further studies are required to elucidate the role of these proteins in GLUT4 endocytosis in IR adipose per se in dairy cows. The expression of ANXA1 was higher in IR than in IS adipose. Several studies have shown altered ANXA1 expression in adipose tissue of obese or diabetic humans; increased ANXA1 expression was found in white adipose tissue of obese humans,27 although the abundance of ANXA1 was reduced in visceral adipose tissue of type 2 diabetic humans.28 ANXA1 is a member of the annexin family of calcium- and phospholipid-binding proteins



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implicated in the regulation of many cellular functions, such as phagocytosis, cell signaling, and proliferation.29 Moreover, these proteins play a pivotal role as regulators of the innate and adaptive immune systems.23 ANXA1 also supports aspects of adipose tissue mass and alters the sensitivity of epididymal adipose tissue to catecholamines and glucocorticoids, thereby modulating lipolysis and IL-6 release.31 As mentioned earlier, ANXA1 is also related to adiposity in cattle.14 Due to the multiple functions of ANXA1 in adipose tissue, further investigations are needed to explain its increased expression in IR adipose of dairy cows. The proteomic analysis in the present study revealed several proteins that can serve as novel biomarkers of IR adipose and of dairy cows' metabolic status. We postulated that cows with IR adipose represent a subgroup of dairy cows, and that these traits might have a genetic basis;8 indeed, the cow-specific repeated pattern of BW loss between lactations (Figure 3) further supports this hypothesis. Similarly, a number of studies in humans have identified a genetic influence on phenotypes associated with metabolic disorders that include multiple measures of adiposity, hyperinsulinemia and IR.32–34

Conclusions

A unique pattern of protein expression was found in dairy cows that have IR adipose tissue during the peripartum period. We identified novel biomarkers of IR adipose that are related to lipid metabolism and possibly GLUT4 endocytosis. Further studies are required to validate the use of these biomarkers to characterize subpopulations of cows that are better adapted to metabolic stress around calving.

Supporting Information, this material is available free of charge via http://pubs.acs.org/. The full list of proteins and raw data are listed in Supplementary Table 1, and Supplementary


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Figure 1 describes a radial display of one of the most relevant networks involving DEP of IR adipose according to IPA®.

ACKNOWLEDGMENTS

The author would like to thank Y. Levin and T. Shalit (INCPM, Weizmann Institute of Science, Rehovot, Israel) for their assistance in proteomic analysis, statistics and bioinformatics analysis, and S. Boura-Halfon (Weizmann Institute of Science, Rehovot) for the insightful advice.

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Mayer, E. J.; Newman, B.; Austin, M. A.; Zhang, D.; Quesenberry, C. P.; Edwards, K.; Selby, J. V. Genetic and environmental influences on insulin levels and the insulin



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Table 1. Cellular Location and Function of Proteins Detected in Adipose Tissue of Dairy Cows Cellular locationa Cytoplasm

Function enzyme kinase other peptidase phosphatase transcription regulator translation regulator transporter

n 91 10 96 13 3 3 5 14

% 38.72 4.26 40.85 5.53 1.28 1.28 2.13 5.96

Extracellular space

cytokine enzyme growth factor other peptidase phosphatase transporter

1 8 3 64 9 1 8

1.02 8.16 3.06 65.31 9.18 1.02 8.16

Nucleus

enzyme kinase other peptidase transcription regulator transporter

9 1 37 3 9 3

14.52 1.61 59.68 4.84 14.52 4.84

Unknown location

enzyme kinase other

2 1 5

25.00 12.50 62.50

Plasma membrane

enzyme ion channel other peptidase transmembrane receptor transporter

9 1 34 2 3 7

16.07 1.79 60.71 3.57 5.36 12.50

a

Based on function analysis by Ingenuity®



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Table 2. The Most Relevant Functions in IR vs. IS Adipose in Dairy Cows Function annotationa

P-value

No. of molecules

Inflammatory response

0.0045

10

Organization of actin cytoskeleton

0.0000

9

Leukocyte migration

0.0040

11

Homing of mononuclear leukocytes

0.0021

5

Proliferation of T lymphocytes

0.0006

10

Reorganization of cytoskeleton

0.0000

7

Accumulation of lipid

0.00005

8

Release of lipid

0.00031

6

Lipolysis of adipose tissue

0.00038

3

Release of cholesterol

0.00093

2

Synthesis of acetyl-coenzyme A

0.00093

2

Lipid-related functions

a

Based on function analysis by Ingenuity®


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Figure legends Figure 1. Most relevant functions that were differentially expressed during late pregnancy and early lactation. Functions were identified by Ingenuity® according to differentially expressed proteins between postpartum and prepartum adipose in dairy cows. Figure 2. Body-weight (BW) and plasma nonesterified fatty acid (NEFA) concentrations in peripartum cows with insulin-resistant (IR, n = 4) or insulin-sensitive (IS, n = 4) adipose. (A) Weekly averages (± SE) of BW of cows with IR adipose (solid line) and those with IS adipose (broken line). (B) Weekly averages of plasma NEFA concentrations (± SE) in cows with IR adipose (solid line) or those with IS adipose (broken line). Figure 3. Repetitive degree of body-weight (BW) loss in the first month postpartum during all lactations (number 1–5) in cows with insulin-resistant (IR, n = 4) or insulin-sensitive (IS, n = 4) adipose. Two cows (one from each subgroup) were in lactation number 3 and one IS cow was in lactation number 4, hence their data was incomplete regarding lactations 4-5. BW loss during the first 30 d postpartum was calculated for each cow during all lactations (1 to 5), and the average BW loss (± SE) between each lactation in cows with IR adipose (black bar) or with IS adipose (gray bar) are presented. Average BW loss during the first 30 d postpartum was higher in cows with IR compared to IS adipose across lactations (P < 0.0079), and also in lactations number 2, 3 and 5 (P < 0.05). Figure 4. Heat map of all differentially expressed proteins between insulin-resistant (IR) and insulin-sensitive (IS) adipose in peripartum dairy cows. The distinctive pattern of protein expression is shown in IR adipose prepartum (lanes 1–4) and postpartum (lanes 5–7), and in IS adipose prepartum (lanes 8–12) and postpartum (lanes 13–16). Figure 5. Most relevant pathways according to differentially expressed proteins in insulinresistant (IR) compared to insulin-sensitive (IS) adipose. Functions were identified by



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Ingenuity® according to differentially expressed proteins between IR and IS adipose in dairy cows. Figure 6. Western-blotting validation of (A) monoglyceride lipase (MGLL) and (B) hormone sensitive lipase (HSL) in prepartum and postpartum insulin-resistant (IR, n = 4) vs. insulinsensitive (IS, n = 4) adipose of dairy cows. Total protein extractions (20 µg) from adipose biopsies from 260-d-pregnant, nonlactating cows (prepartum) and cows 4 d after calving (postpartum) were resolved by SDS-PAGE. Proteins were immuno-blotted with HSL and MGLL antibodies. Densitometry analysis of 8 cows (mean ± SEM) are presented as bar graphs. The abundance of MGLL was higher in IR than in IS adipose (P < 0.02), and the abundance of HSL prepartum was numerically higher in IR than in IS adipose (P < 0.2), whereas postpartum it was similar between IR and in IS adipose, which is in accordance with the proteomic analysis (see text). Supplementary Figure 1. One of the most relevant networks according to IPA (Ingenuity®) related to lipid metabolism in IR adipose.


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For TOC only



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Figure 1. Most relevant functions that were differentially expressed during late pregnancy and early lactation. Functions were identified by Ingenuity® according to differentially expressed proteins between postpartum and prepartum adipose in dairy cows. 177x228mm (150 x 150 DPI)

ACS Paragon Plus Environment

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Figure 2. Body-weight (BW) and plasma nonesterified fatty acid (NEFA) concentrations in peripartum cows with insulin-resistant (IR, n = 4) or insulin-sensitive (IS, n = 4) adipose. (A) Weekly averages (± SE) of BW of cows with IR adipose (solid line) and those with IS adipose (broken line). (B) Weekly averages of plasma NEFA concentrations (± SE) in cows with IR adipose (solid line) or those with IS adipose (broken line). 104x229mm (150 x 150 DPI)



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Figure 3. Repetitive degree of body-weight (BW) loss in the first month postpartum during all lactations (number 1–5) in cows with insulin-resistant (IR, n = 4) or insulin-sensitive (IS, n = 4) adipose. Two cows (one from each subgroup) were in lactation number 3 and one IS cow was in lactation number 4, hence their data was incomplete regarding lactations 4-5. BW loss during the first 30 d postpartum was calculated for each cow during all lactations (1 to 5), and the average BW loss (± SE) between each lactation in cows with IR adipose (black bar) or with IS adipose (gray bar) are presented. Average BW loss during the first 30 d postpartum was higher in cows with IR compared to IS adipose across lactations (P < 0.0079), and also in lactations number 2, 3 and 5 (P < 0.05). 178x228mm (150 x 150 DPI)


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Figure 4. Heat map of all differentially expressed proteins between insulin-resistant (IR) and insulinsensitive (IS) adipose in peripartum dairy cows. The distinctive pattern of protein expression is shown in IR adipose prepartum (lanes 1–4) and postpartum (lanes 5–7), and in IS adipose prepartum (lanes 8–12) and postpartum (lanes 13–16). 177x228mm (140 x 67 DPI)



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Figure 5. Most relevant pathways according to differentially expressed proteins in insulin-resistant (IR) compared to insulin-sensitive (IS) adipose. Functions were identified by Ingenuity® according to differentially expressed proteins between IR and IS adipose in dairy cows. 177x228mm (150 x 150 DPI)


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Figure 6. Western-blotting validation of (A) monoglyceride lipase (MGLL) and (B) hormone sensitive lipase (HSL) in prepartum and postpartum insulin-resistant (IR, n = 4) vs. insulin-sensitive (IS, n = 4) adipose of dairy cows. Total protein extractions (20 µg) from adipose biopsies from 260-d-pregnant, nonlactating cows (prepartum) and cows 4 d after calving (postpartum) were resolved by SDS-PAGE. Proteins were immunoblotted with HSL and MGLL antibodies. Densitometry analysis of 8 cows (mean ± SEM) are presented as bar graphs. The abundance of MGLL was higher in IR than in IS adipose (P < 0.02), and the abundance of HSL prepartum was numerically higher in IR than in IS adipose (P < 0.2), whereas postpartum it was similar between IR and in IS adipose, which is in accordance with the proteomic analysis (see text). 138x220mm (150 x 150 DPI)


Defining the Adipose Tissue Proteome of Dairy Cows to Reveal

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