involvement of skeletal muscle protein, glycogen, and fat metabolism in the adaptation on early...

Published: July 21, 2011

r 2011 American Chemical Society 4252 dx.doi.org/10.1021/pr200425h | J. Proteome Res. 2011, 10, 4252–4262

ARTICLE

pubs.acs.org/jpr

Involvement of Skeletal Muscle Protein, Glycogen, and FatMetabolism in the Adaptation on Early Lactation of Dairy CowsBj€orn Kuhla,*,† Gerd N€urnberg,‡ Dirk Albrecht,§ Solvig G€ors,† Harald M. Hammon,† andCornelia C. Metges†

†Research Unit Nutritional Physiology “Oskar Kellner”, Leibniz Institute for Farm Animal Biology (FBN), Wilhelm-Stahl-Allee 2,18196 Dummerstorf, Germany‡Research Unit Genetics and Biometry, Leibniz Institute for Farm Animal Biology (FBN), Wilhelm-Stahl-Allee 2, 18196 Dummerstorf,Germany§Institute of Microbiology, Ernst-Moritz-Arndt-University, F.-L.-Jahn-Strasse 15, 17487 Greifswald, Germany

bS Supporting Information

’ INTRODUCTION

In the last days of gestation, dairy cows begin to reduce theirfeed intake until parturition and only slowly increase feed intaketo reach a maximum by the sixth week postpartum. Thus, high-yielding dairy cows ingest less nutrients and energy than theyrequire for meeting energy demands of milk production duringearly lactation.1 As a consequence, animals experience negativeenergy balance in which they mobilize their fat, glycogen, andprotein reserves. The release of body reserves particular that offatty acids but also amino acids into circulation has been sug-gested to enable a high level of milk production but also toprevent sufficient feed intake in the early postparturient period.1

The mobilization of body protein in early lactation may amountto more than 20 kg in dairy cows.2�4 Much of this mobilizedprotein appears to be derived from peripheral tissues, primarilyskeletal muscle5 and, to a lesser extent, skin, through suppressionof tissue protein synthesis, and increased proteolysis.6,7 Thereleased amino acids are intensively used for milk protein syn-thesis, direct oxidation, or gluconeogenesis but presumably to adifferent extent. As a result, this might cause an imbalanced

amino acid pattern in the circulation including the enrichment ofcertain amino acids.5,8,9 But although numerous amino acidsexert highest satiating effects when compared with other macro-nutrients,10 the role of amino acids released from muscle as wellas muscle metabolic processes potentially involved in regulatingenergy balance and feed intake during early lactation is stillunknown.

Within the circuits controlling feed intake, two major afferentpathways integrate protein and amino acid signals at the brainlevel: the indirect nervous-mediated and the direct blood path-way.10 Within the latter, the muscle as the predominant site ofprotein storage may undergo proteolysis around parturition andthereby releases creatinine, 3-methyl histidine,11,12 and otheramino acids into circulation.5 Only recently has it been shownthat elevated concentrations of leucine for example influence nut-rient derived signaling in the central nervous system and reducesfood intake.13 In addition, excess of specific amino acids, including

Received: May 9, 2011

ABSTRACT: During early lactation, high-yielding dairy cowscannot consume enough feed tomeet nutrient requirements. Asa consequence, animals drop into negative energy balance andmobilize body reserves including muscle protein and glycogenfor milk production, direct oxidation, and hepatic gluconeogen-esis. To examine which muscle metabolic processes contributeto the adaptation during early lactation, six German Holsteincows were blood sampled and muscle biopsied throughout theperiparturient period. Frompregnancy to lactation, the free plasmaamino acid pattern imbalanced and plasma glucose decreased.Several muscle amino acids, as well as total muscle protein, fat, andglycogen, and the expression of glucose transporter-4were reducedwithin the first 4 weeks of lactation. The 2-DE and MALDI-TOF-MS analysis identified 43 differentially expressed muscle protein spotsthroughout the periparturient period. In early lactation, expression of cytoskeletal proteins and enzymes involved in glycogen synthesis andin the TCA cycle was decreased, whereas proteins related to glycolysis, fatty acid degradation, lactate, and ATP productionwere increased.On the basis of these results, we propose amodel in which themuscle breakdown in early lactation provides substrates formilk productionby a decoupled Cori cycle favoring hepatic gluconeogenesis and by interfering with feed intake signaling.

KEYWORDS: muscle, dairy cow, parturition, two-dimensional gel electrophoresis, mass spectrometry

4253 dx.doi.org/10.1021/pr200425h |J. Proteome Res. 2011, 10, 4252–4262

Journal of Proteome Research ARTICLE

Met, Trp, and His, or increase in blood NH3 can exert toxiceffects associated with aversive anorexic responses.10,14 More-over, the muscle may release various myokines such as inter-leukin-6, interleukin-15, leukemia inhibitory factor (LIF), orbrain-derived neurotrophic factor (BNDF) into the bloodstreamto modulate energy metabolism and food intake via the directpathway.15

The nervous-mediated pathway is also involved in the regula-tion of feed intake. For example, while inhibition of hepatic fattyacid oxidation alone does not influence feeding behavior, addi-tional suppression of fatty acid oxidation in muscle stimulatesfood intake,16 suggesting the involvement of muscle metabolismin the control of feed intake. Also, ablation of nerves innervatingskeletal muscles blocked the ability of intrahypothalamic injectedleptin to stimulate fatty acid oxidation in the muscle.17 This effectis mediated via the R-adrenergic pathway resulting in theactivation of muscle 50-AMP-activated kinase (AMPK)—a keyregulator of myocyte energy status.17

However, it is largely unknown how muscle proteolysis affectscirculating amino acid concentrations and which metabolic path-ways are activated or deactivated throughout the periparturientperiod. In order to identify these signals andmetabolic events, weperformed muscle amino acid profiling and proteome analysis toprovide information on both amino acid release (direct pathway)as well as muscle metabolism (indirect pathway).

’METHODS

Animals and BiopsiesSix German Holstein cows in first and second lactation were

ceased to be milked 9 weeks before expected calving (ages at thattime: mean( SD = 44( 5 month) and were fed a dry-off rationbased on grass silage. With the beginning of the close-up periodstarting from 30 days before expected parturition and duringlactation cows received ad libitum a total mixed ration (TMR)twice daily (4.30 a.m. and 11.30 a.m.). TMR consisted of cornand grass silage, hay and concentrate (6.4MJ net energy lactation/kg dry matter (NEL/kg DM) for the last 30 days of gestation(close-up period) and 7.0MJ NEL/kg DM for lactation. NEL wascalculated according to the German Society of Nutrition Physio-logy.18 Feed intake was recorded daily. During lactation, cowswere milked twice daily (4.00 a.m. and 3.30 p.m.). Energycorrected milk yield increased to 50.7 kg/d by the fourth weekpostpartum.

The semitendinosus muscles were biopsied after the morningmilking and feeding, alternating left and right at days �24, +1,+14 and +28 (�3, 0, +2, +4 weeks) relative to their second/3rdparturition using a custom-made biopsy shooting apparatus. Dueto antibiotic therapy, one cow was not biospied at +1 day afterparturition. The treatment was in accordance with the guidelinesfor the use of animals as experimental subjects of the State Gov-ernment in Mecklenburg-West Pommerania (Registration No.LALLF M-V/TSD/7221.3�2.1�019/08).

Sample Preparation for 2-DE AnalysesThe biopsy (∼0.5 mm � 3 cm; approximately 500 mg) was

liberated from skin and subcutaneous fat and muscle tissue wasshock-frozen in liquid N2. Frozen muscle tissue was crushed to afine powder in a mortar under liquid N2. Tissue powder (50 mg)was homogenized using a Teflon pestle in 200 μL of 8 M urea,50 mM Tris, 2% CHAPS, 40 mM DTT, 0.5% IPG-buffer (allfrom Amersham Biosciences, Uppsala, Sweden). After centrifugation

(11 000� g, 4 �C, 20 min), the protein concentration in thesupernatant was measured according to the Bradford methodusing BSA as standard.

2-D ElectrophoresisIndividual muscle samples (n = 23) were run in technical

duplets yielding 46 gels in total. A sample of 500 μg protein wasadded to 320 μL rehydration buffer (8M urea, 2% CHAPS, 0.8%IPG-buffer, 18 mM DTT and a trace of bromophenol blue),mixed and loaded to 18 cm IPG (pH 3�10) (Amersham Bio-sciences, Uppsala Sweden). After rehydration and IEF, the IPGwas equilibrated in buffer containing 50 mM Tris (pH 8.8), 30%glycerol, 6 M urea, 2% SDS, 1%DTT and than in the same bufferwithout DTT but 2.5% iodoacetamide, each for 15 min. IPGswere transferred to 12.5% SDS PAGE gels (20 � 20 � 0.1 cm)and embedded in low melting agarose. The gels were stainedovernight in colloidal Rotiblue (Roth, Karlsruhe, Germany),destained 3-times in 15% methanol and 5% acetic acid and 1�in distilled water.

Image AnalysisGels were scanned using an Epson Perfection 1250 scanner

and saved as tiff format (8-bit gray scale). The 2-DE imageanalysis was carried out on a computer using Delta2D softwareversion 4.0 (DECODON, Greifswald, Germany; http://www.decodon.com). Gels derived from one animal were warped acc-ording to the “all to one” warping strategy. A fusion image wascreated from all warped images containing all spots from all gels.After automatic spot detection on the fusion image, spot boun-daries were transferred to the original images and there quanti-fied using the gray value of each spot to obtain the spot volume.Each spot volume was normalized to the total spot volume ofeach gel image (=100%) yielding the normalized spot volume in% which was further used for statistical analysis (see below).

Mass SpectrometryProtein identification was performed by the method compiled

previously.19 Briefly, protein spots were punched out using anEttan spot cutter (Amersham) with a 2 mm picker head. Spotswere transferred into 96 well micro titer plates, tryptic digestedand subsequently spotted on a MALDI-target. The molecularmasses of tryptic digests were measured on a 4800 MALDITOF/TOF Analyzer (Applied Biosystems). The spectra wererecorded in a mass range from 900 to 3700 Da with a focus on2000Da. For onemain spectrum, 30 subspectra with 60 shots persubspectrum were accumulated. When the autolytical fragmentsof trypsin with (M +H)+m/z at 1045.556 and 2211.104 reacheda signal-to-noise ratio (S/N) of at least 20, an internal calibrationwas automatically performed as two-point-calibration. The stan-dard mass deviation was less than 0.15 Da. After calibration thepeak lists were created by using the “peak to mascot” script of the4000 Series Explorer Software (V3.5). Selected settings were:mass range from 900 to 3800 Da, peak density of 15 peaks per200 Da, minimal area of 100 and maximal 60 peaks per spot. Thepeak list was created for an S/N ratio of 10.

To confirm the results obtained by MALDI-TOF-MS, MAL-DI-TOF-TOF analysis was on the 4800 MALDI TOF/TOFAnalyzer (Applied Biosystems). The three strongest peaks of theTOF-spectra were selected automatically and measured. For onemain spectrum 25 subspectra with 125 shots per subspectrumwere accumulated using a random search pattern. The internalcalibration was automatically performed as one-point-calibrationwith (M+H)+ m/z at 175.119 or with lysine (M + H)+ m/z at



Table 1. Complete List of Differentially Expressed Proteins (ANOVA p < 0.05; Tukey test p < 0.1) and Affiliation to ClusterAnalysis Displayed in Figure 5

spot numbera protein name accession number cluster Tukey test p < 0.1 between weeks rel. to parturition

Cytoskeletal proteins

169 R-actin 56204817 1 0 vs 4; 2 vs 4

171 R-actin 56204817 1 �3 vs 0; 3 vs 0; 2 vs 4

16 R-actin 56204817 1 0 vs 4; 2 vs 4

55 R-actin 56204817 2 �3 vs 2

167 actin in complex with Kabiramide C 39654752 9 �3 vs 0; �3 vs 2; �3 vs 4

172 complex between actin: Gelsolin Domain 1 7766848 1 0 vs 4; 2 vs 4

175 R-Crystallin, B chain 57085977 1 �3 vs 0; 0 vs 4; 0 vs 2

164 myosin-1 (MYO1) 41386691 2 �3 vs 2; �3 vs 0

165 myosin-1 (MYO1) 41386691 9 �3 vs 0; �3 vs 4

63 Myotilin (MYOT) 116734833 8 �3 vs 4; 0 vs 4

186 troponin T, beta isoform (TNNT) 259912 4 0 vs 4

159 bridging integrator 1 (Bin1) 114052603 1 0 vs 4

117 PDZ and LIM domain protein 3 (PDLIM3) 77736235 6 �3 vs 2; 0 vs 2

190 PDZ and LIM domain protein 3 (PDLIM3) 77736235 6 0 vs 2

153 vimentin (VIM) 110347570 1 0 vs 4; 2 vs 4

Glycolysis and Glycogenesis

51 glyceraldehyde-3-phosphate dehydrogenase 163866419 7 0 vs 4

174 glyceraldehyde-3-phosphate dehydrogenase 77404273 8 �3 vs 4; 0 vs 4

124 glyceraldehyde-3-phosphate dehydrogenase 40889050 1 0 vs 4

173 glyceraldehyde-3-phosphate dehydrogenase 77404273 4 0 vs 2; 0 vs 4

168 glyceraldehyde-phosphate-dehydrogenase 53680576 7 �3 vs 0; 0 vs 2

35 fructose-bisphosphate aldolase A (ALDOA) 156120479 6 �3 vs 2; �3 vs 4

110 pyruvate kinase (PK) 194670470 6 �3 vs 2

61 triosephosphate isomerase (TPI) 61888856 5 0 vs 2

59 R-enolase (ENOA) 73956728 1 0 vs 4; 2 vs 4

46 L-lactate dehydrogenase, A chain (LDH-A) 119920080 6 �3 vs 2

19 UTP-glucose-1-phosphate uridylyltransferase (UGP1) 41386780 3 0 vs 2

TCA cycle and ATP homeostasis

1 aconitase 2 (ACO2) 74268076 8 �3 vs 4; 0 vs 4; 2 vs 4

163 aconitase 2 (ACO2 90970312 8 2 vs 4

194 malate dehydrogenase (MDH) 77736203 4 0 vs 4

193 creatine kinase, M chain (CK) 4838363 9 �3 vs 2

152 creatine kinase, M chain (CK) 4838363 10 2 vs 4

118 creatine kinase, M chain (CK) 4838363 6 �3 vs 2; 0 vs 2; 2 vs 4

45 ATP synthase, subunit alpha (ATP5A) 27807237 7 �3 vs 0

150 adenylate kinase 1 (AK1) 61888850 3 0 vs 2

Fatty acid oxidation

23 retinal dehydrogenase 1 (RALDH 1) 160332357 5 0 vs 2

38 electron transfer flavoprotein, subunit alpha (ETFA) 115496196 10 2 vs 4

Protein metabolism

39 Dj-1 protein 33358055 3 �3 vs 2; 0 vs 2

86 phosphatidylethanolamine-binding protein 1 (PEBP1) 75812940 7 �3 vs 0; �3 vs 2; �3 vs 4

71 heat shock protein beta-1 (HSPB1) 71037405 10 2 vs 4

73 heat shock protein beta-1 (HSPB1) 71037405 8 �3 vs 4

Binding and transport

4 serum albumin (ALB) 1351907 1 0 vs 4

134 Myoglobin (MB) 73586735 6 �3 vs 2

133 Myoglobin (MB) 215512088 6 �3 vs 2; 0 vs 2; 2 vs 4a Spot number refers to Supplementary Figure 1, Supporting Information.



147.107 reached a signal-to-noise ratio (S/N) of at least 5.The peak lists were created as described above for a S/N ratioof 7. Selected settings were: mass range from 60 to precursor�20 Da, peak density of 15 peaks per 200 Da, minimal area of100 and maximal 65 peaks per precursor.

For the identification of proteins, database search with PMF ofthe analyte was performed against the databasesNCBInr (NationalCenter for Biotechnology Information, http://www.ncbi.nlm.nih.gov/) and Swiss-Prot (http://us.expasy.org/sprot/) using theMascot search engine version 2.1 (Matrix Science Ltd., London,U.K.). Search parameters were taxonomy: “all entries”; variablemodifications: “carbamidomethyl (C)” and “oxidation (M)”; pep-tide tolerance (50 ppm; peptide charge “1+”; MS/MS tolerance“0.5 Da”; “monoisotopic”.

Determination ofMuscle Protein, Fat andGlycogen ContentTissue powder (50 mg) was dried in a muffle type furnace at

105 �C for 3 h to determine content of dry matter (DM). Driedsamples were analyzed for nitrogen and carbon content at anelement CNS � 2000 analyzer (LECO Instrumente GmbH,M€onchengladbach, Germany). Fat and protein content werecalculated according to the method described previously.20 Forthe analysis of glycogen, 25 mg wet tissue was applied to anenzyme-based starch kit (#10207748035; Boehringer Man-nheim, Germany) according to the manufacture’s introduction.

Western Blot AnalysisDue to the limited amount of biopsy material powdered

muscle tissue (20 mg) from only three animals was extractedin 60 μL lysis buffer (50 mM Tris buffered saline (TBS; pH 7.6)containing 1 mM EDTA, 100 mM NaF, 1 mM NaVO4, 0.5%DOC, 0.1% SDS, 1% Igepal). Extracts were centrifuged at13.000 rpm for 10 min at 4 �C. Protein extracts were treatedwith Laemmli buffer, boiled for 5 min, loaded on a 12% SDS gel,and electrotransferred to nitrocellulose. Blots were reversiblystained with Ponceau S visualizing total proteins, washed andincubated with rabbit anti GLUT4 IgG (each 1:750; Biotrend,Germany) for 16 h at 4 �C. After 5 washing steps in TBST (TBScontaining 0.05% Tween 20), horseradish peroxidase-labeledanti rabbit antibody (1:10 000; Santa Cruz, Santa Cruz, CA) wasapplied for 2 h at room temperature. After washing, blots weredeveloped on hyperfilmes using Enhanced Chemiluminescence(ECL) reaction. Hyperfilms and Ponceau stained blots werescanned and digital images were analyzed by ImageJ software.

Glucose, Urea and Amino Acid AnalysesBlood was collected from the jugular vein at day�24,�7, +1,

+7, +15, +28 (�3,�1, 0, +1, +2, +4 weeks) relative to parturitionin sodium EDTA tubes (Sarstedt AG & CO, N€umbrecht,Germany). Blood samples were centrifuged for 20 min at 4 �Cand 2000� g and the plasma frozen at�80 �C for later analyses.Plasma glucose and urea were analyzed by kits LT GL 0251 (forglucose) and LT UR 0010 (for urea) purchased from Labor andTechnik Lehmann (Berlin, Germany). All measurements wereperformed by an automatic analyzer (Cobas Mira Plus; Roche,Basel, Switzerland).

For amino acids analyses, plasma samples were diluted withwater (1:10) and free amino acids (FAA) were analyzed byHPLCequipped with a fluorescence detector (Series 1200; AgilentTechnologies, Waldbronn, Germany) as described previously.19

Therein, precolumn derivatization was performed with ortho-phthalaldehyde (OPA)/3-mercaptopropionic acid (MCPA) forprimary and 9-fluorenylmethoxycarbonyl chloride (FMOC) forsecondary amino acids using MCPA as reducing agent andiodoacetic acid to block sulfhydryl groups. Standard mixtures ofamino acids (completed with asparagine and glutamine) allowedassignment of retention times and quantification.

For the analysis ofmuscle amino acids (free and proteinogenic),5 mg of powdered tissue was treated with 2 mL 6 N HCl andincubated at 110 �C for 24 h. The hydrolysate was dried at 60 �Cunder N2 atmosphere, dissolved in 4 mL water, and subjected toHPLC as described above.

StatisticsFor statistical evaluation of feed intake, intermediary plasma

metabolites, and muscle amino acids, glycogen and total fat, aswell as protein expression pattern, an one-way repeated measuresANOVA analysis was calculated with repeated factor time (e.g.,for biopsies: �3, 0, +2, +4 weeks relative to parturition; seeabove). As posthoc test, we used Tukey’s multiple comparisontest for pairwise differences between time points to ensuremultiple comparison adjustment for the p-values.

For evaluation of the protein expression time course, we onlyconsidered those protein spots from the repeated measureANOVA analysis with at least one significant expression differ-ence between two time points (Tukey adjusted p-value < 0.10;see Table 1). Following the aim to group proteins with a similarexpression—time course, we rescaled the normalized spot vol-ume values obtained from Image analysis (see above) at timepoint 0, +2, +4 (postpartum) relative to time point �3 (antepartum). To this end, the expression value at time point �3 wasset equal to 1 and the expression values at time point 0, +2, +4were divided by the expression value at time point �3. By thisway, one gets more comparable, relative expression courses in-dependently of the gray value-based expression scale. With theserelative expression values, we conducted a cluster analysis usingproc VARCLUS of SAS (2009).21 After clustering, means of therelative expression values of those spots forming one cluster werecalculated (see Figure 5).

’RESULTS

DM Intake and Plasma Amino Acid Changes in EarlyLactation

DM intake continuously declined starting 4 weeks beforeparturition until calving to 81% (P < 0.01) and increased until the

Figure 1. Average daily DM intake of dairy cows ranging between week4 a.p. and week 5 p.p. The value at week “0” represents the DM intake atthe day of calving. Data are presented as mean( SEM. The gray arrowsillustrate the day of muscle biopsy sampling. DM intake significantlydiffered over time at P < 0.001 (ANOVA).When compared to the day ofcalving, feed intake between week +2 and +5 relative to parturitionsignificantly differed (*) at P < 0.05 (Tukey-test).



fifth week after parturition gaining 163% in comparison to theintake at calving (P < 0.001; ANOVA) (Figure 1).

According to the course of DM intake, plasma concentrationsof free essential amino acids (EAA) - except of His and Met �show a nadir at the first day after calving (Figure 2). His, Met andthe nonessential amino acids (NEAA) Asp, Gln, Glu, Cys, Tyr,and Orn as well as plasma glucose continuously decrease untilcalving and remain lower in early lactation than days a.p. (P <0.05, ANOVA). In contrast, concentrations of Gly, Tau, andR-aminobutyric acid (R-ABA) continuously increase startingeither one week before or at calving until the fourth week afterp.p. and thereby exceeding the level observed prior to parturition(P < 0.05, ANOVA). Mean concentrations of Asn, Ser, Pro,Hy-Pro, and Me-His, peak either at or in the first week oflactation but only the latter two reach significance level (P <0.05, ANOVA).

Muscle Amino Acid, Glycogen and Fat in Early LactationTo examine which amino acids are primarily released by the

muscle during early lactation, tissue specimens were hydrolyzed

to release free and proteinogenic amino acids characterizing theamino acid profile in the muscle. In the fourth week p.p., muscleEAA, Gln/Glu, Asp/Asn, Tyr, and Ser are reduced to ∼86%when compared to the third week a.p. (Figure 3). Whereas mus-cle Pro, His, Tau, β-Ala, and Me-His levels did not change overtime (P > 0.1), Gly levels (P < 0.07) are lowest at parturition(Figure 3). Likewise, muscle glycogen and total fat content reacha nadir at 2 weeks p.p. whereas total muscle protein tends todecrease after parturition (Figure 4).

Metabolic Changes in Early LactationIn order to elucidate cellular and metabolic processes under-

lying the mobilization of energy reserves stored in muscle tissue,we performed a 2D-GE based proteome analysis. Out of 601spots detected at the gel image, we picked all clearly separatedand visually recognizable spots (n = 250) and among themidentified 181 spots by MALDI-TOF mass spectrometry (Sup-plementary Figure 1, Supplementary Table 1, Supporting In-formation). Among them, we found 75 spots of cytoskeletalorigin, 14 spots with binding and transport properties, 20 spots

Figure 2. Periparturient plasma amino acid, glucose and urea concentrations of six dairy cows. In the first row essential amino acids, in the second andthird row nonessential amino acids are displayed. Data are presented as mean ( SEM. Differences between the ante partum and postpartum periodaccording to P < 0.05 are marked by * whereas P < 0.1 is indicated by # (Tukey-test).



referring to protein and amino acid metabolism, 48 spotsrepresenting enzymes involved in glycolysis and TCA cycle, 15spots referring to pH and ATP homeostasis, and 9 spots withmiscellaneous function including regulators of fat metabolism(Supplemental Table 1, Supporting Information). Statisticalanalyses revealed that 43 of the identified 181 spots are differ-entially regulated throughout the periparturient period (Table 1).These 43 spots were assigned to 10 clusters according to a similarexpression-time course (Figure 5).

In early lactation, there is increased expression of glycolyticenzymes such as fructose bisphosphate aldolase A, GAPDH,triosephosphate isomerase, enolase, pyruvate kinase and lactatedehydrogenase (123�198%) as compared to the third week a.p.Conversely, we found decreased glycogenesis as indicated by thelowest expression of UTP-glucose-1-phosphate uridylyltransfer-ase (∼55%) 2 weeks p.p. Furthermore, expression of TCA cycleenzymes (aconitase 2, malate dehydrogenase; MDH) is alsolowered (54�74%) 4 weeks p.p. whereas expression of enzymesregulating ATP homeostasis such as creatine kinase (CK) andATP synthase are upregulated (125�185%) as compared to latepregnancy. Two proteins closely associated with fatty acid degra-dation namely electron transfer flavoprotein (ETF) and retinaldehydrogenase 1 (RALDH1) are also increasingly expressed(196 and 162%, repectively) in early lactation.

Furthermore, we observed coordinated downregulation ofproteins involved in protein synthesis and stabilization (Dj-1protein, heat shock protein beta-1 (HSBP1) and its bindingpartner R-crystallin as well as upregulation of the cell growth

suppressor phosphatidylethanolamine-binding protein 1 (PEBP1)in early lactation.

During this period, a number of cytoskeletal and structuralproteins such as R-actin, myosin, myotilin, troponin, vimentin,and R-crystallin were also decreased (64�41%). The abundanceof the transport protein myoglobin peaked in the second weekp.p. whereas that of serum albumin was highest 4 weeks p.p.

Figure 3. Amino acid composition of muscle hydrolysates during the periparturient period. In the first row essential amino acids, in the second and thirdrow nonessential amino acids are displayed. Due to hydrolysis, Gln and Asn are determined as Glu and Asp, respectively, whereas R-ABA, Cit, Car, Trp,and Orn were not detected. Data are presented as mean( SEM. The asterisks indicate differences at Pe 0.05 between the ante partum and postpartumperiod whereas # shows differences with P < 0.1 (Tukey-test).

Figure 4. Muscle glycogen, fat and protein content of cows (% wetweight) during the periparturient period. Data are presented as mean +SEM and statistical differences for ANOVA are displayed. Pair-wisecomparisons indicated by * are based on P < 0.05 (Tukey test).



GLUT4 Expression in Early LactationTo examine whether increased muscle glycolytic processes

described above are supplied by glycogen degradation or in-creased glucose uptake from the circulation, we examined theexpression of the glucose transporter 4 (GLUT4) in muscle

tissue. GLUT4 is insulin-regulated and due to the insulin defi-ciency expected to be altered in early lactation. GLUT4 Westernblot analysis was performed—similar to the proteome imageanalysis—by normalization to the total protein load stained withPonceau (∼20�250 kDa), because cytoskeletal proteins as wellas GAPDH potentially serving as housekeeping genes wereregulated (see above). We found that relative expression ofGLUT4 is reduced to∼60% in the fourth week p.p. as comparedto the level around parturition (Figure 6).

’DISCUSSION

Plasma AABecause of the continuous outflow of microbial and unde-

graded dietary protein from the rumen, absorption and periph-eral plasma concentrations in dairy cows are relatively unchangedduring the day and thus unaffected by variation in time after thelast milking or meal intake.22 During late gestation, however, fetalgrowth, lactogenesis and during early lactation milk protein syn-thesis but also gluconeogenesis require enormous amounts ofcertain AA that are withdrawn from the circulation resulting in animbalanced AA pattern in the blood. This can be seen in mostplasma FAA falling immediately after parturition. Another reasonfor decreasing plasma AAmay be the reduced DM intake aroundparturition that reduces the supply from the intestinal tractand thus additionally contributes to an imbalanced plasma AApattern. However, increased DM intake after the second week oflactation supports the recovery of the concentration of most ofthe FAA felt around parturition. Our results are in agreementwith earlier studies,5,8,9 which reported FAA concentrations overa longer postparturient period.

The plasma concentrations of total NEAA, primarily those ofplasma Tau, Ser, Asn, Gly, Hy-Pro, R-ABA, and Me-His increaseafter parturition and remain elevated as compared to the a.p.period, suggesting that body protein is degraded. This degrada-tion is directed to counteract the circulating AA imbalance which

Figure 5. Relative expression patterns of 43 differentially expressed protein spots obtained using cluster analysis. Fold-change in expression relative toweek �3 is displayed within 10 clusters. See at the right-hand for proteins within clusters and in Table 1 for listened protein spots.

Figure 6. Western Blot analysis of muscle GLUT4 in three periparturi-ent cows. Tissue extracts underwent electrophoresis and blotting. Blotswere stained by Ponceau and subsequent immunoblotted for GLUT4.For densitometrical analysis GLUT4 immunoreactivity was normalizedto the total protein content of the blot as stained by Ponceau. Fold-change of expression relative to week �3 was calculated and data areshown as mean + SEM. Pair-wise comparisons are indicated by * (Tukeytest; P < 0.05).



in turn is driven by withdrawal of amino acids for gluconeogen-esis and milk protein synthesis. For example, when the contribu-tion of Ala increase to as much as 5% for hepatic gluconeogenesisin early lactation,23 plasma Ala does not fall suggesting that wholebody protein degradation copes with the increased demand ofglucogenic precursors. Other amino acids mobilized from bodyprotein in early lactation are mainly directed to milk proteinsynthesis24 leading to a deficiency of certain AA in the circulation.On the other hand, mobilized AA that are not used for syntheticprocesses and exceed oxidative capacity may accumulate inplasma and thus contribute to AA imbalance. For example,Me-His, which occurs only in actin and myosin accumulates inearly lactation and has been suggested to predict milk proteinyield.11 Interestingly,R-ABA has been shown to be released fromforearm muscle tissue after short-term starvation in man25 but ithas not been analyzed in altered metabolic conditions of cows sofar. Here we describe for the first time that R-ABA is 2.5-timeselevated in early lactation but not after feed restriction for 60 h;26

however, increased R-ABA may also be generated by accele-rated conversion of cystathionine in the liver.27 Cystathionineis produced from Met metabolism and interestingly, Met con-centrations declined after calving. Also, the ratio of R-ABA/cystathionine may predict fatty liver disease in humans27,28 anddairy cows similarly show signs of increased liver fat content inearly lactation.1

Muscle AAThe amount of FAA in the muscle reflects the largest part of

the FAA pool in the body and was previously analyzed in peri-parturient cows.5 However, changes in FAA in the intracellularmuscle fluid do not necessarily reflect muscle tissue breakdown.Hence, wemeasured total (free and proteinogenic) AA inmusclebiopsies before and after parturition. With the exception of Pro,His, Tau, Gly, β-Ala, and Me-His, all muscle AA determined butalso the total protein content decreased p.p. indicating a pro-nounced negative N balance. This finding is not in contrast to an

earlier result describing increasing muscle FAA concentrationsfrom late pregnancy to lactation.5 Rather our findings suggestthat degradation of muscle proteins contributes to these accumu-lated FAA in muscle during early lactation. Just recently, variousmuscle protein degradation systems including the caspase, theCa2+-dependent, and the ATP-dependent ubiquitin-mediated systemwere found to be activated in early lactation and thus likely accountfor the muscle protein breakdown6 (see also below).

Muscle Ala levels fall only numerically by the fourth week oflactation suggesting only a minor role of nitrogen transport fromthe muscle to the liver within the glucose-alanine cycle. Also,because muscle Gly and Me-His levels are not reduced in theearly postparturient period, it appears that increased plasma Glyand Me-His concentrations in early lactation are not of semi-tendinosus muscle origin.

While muscle protein and amino acid losses continuouslyprogress within the first weeks of lactation, muscle glycogen andfat storages are already exhausted immediately after parturition.This observation points to an early allocation of glucose and fattyacids and a latter allocation of AA either directly for milkproduction or hepatic anabolic processes in early lactation. Insummary, skeletal muscle tissue contributes to the adaptation toearly lactation by mobilizing tissue constitutes in the order ofglycogen, fat and protein.

Muscle Glycolysis, TCA Cycle, and ATP HomeostasisMuscle glycogen degradation likely results in the activation of

the glycolysis pathway as indicated by peaking expression of fiveglycogenolytic and glycolytic enzymes and decreased expressionof one glycogen synthesis enzyme within the first two weeks oflactation. Additionally, we observed highest expression of lactatedehydrogenase in the second week of lactation and continuouslyfalling levels of TCA cycle enzymes by the fourth week of lac-tation, suggesting that (1) pyruvate is shunted toward lactate(rather than toward AcCoA) and (2) ATP production throughthe TCA cycle is decreased. Increased skeletal muscle lactate

Figure 7. Schematic representation of the interaction between protein expression patterns in muscle and physiological events in liver and mammarygland during early lactation. We propose that (1) released amino acids primarily from cytoskeletal degradation are used by the liver as glucoplasticprecursors or by the mammary gland for milk protein synthesis and free AA. (2) Muscle glycogen is used in the glycolysis pathway yielding pyruvatewhich is converted under anaerobic conditions toward lactate. Lactate serves again as precursor for hepatic gluconeogenesis. Because of thereduced GLUT4 expression in muscle and the high glucose demand by the mammary gland, hypoglycaemia prevails resulting in a decoupled Cori cyclein early lactation. Since pyruvate is not converted to AcCoA, the activity of the TCA cycle is diminished and as a compensatory mechanism ATP isproduced via CK and ATP synthase. (3) Stimulated fatty acid degradation is associated with an exhausted (intramuscular) fat content in early lactation.This catabolic event but also elevated PEBP1 signal via the nerval pathway toward feed intake regulatory centers in the brain for modulating feed intake.Hypoglycaemia and imbalanced AA concentrations stimulate feed intake via the humoral pathway.



production presumably serves as substrate for hepatic gluconeo-genesis. Its contribution may reach up to 15�25% in earlylactation.23 Considering the hypoglycaemic conditions, the prio-ritized glucose partitioning to the mammary gland, together withthe reduced expression of muscle GLUT4, we conclude thatduring early lactation the Cori cycle involving glycolytic muscle isdecoupled (Figure 7). However, since the expression of GLUT4in bovine glycolytic muscle is lower than in more oxidativemuscle,29 GLUT4 in oxidative muscle might compensate for thereduced GLUT4 expression in glycolytic muscle. Moreover,beside the insulin-regulated GLUT4, the insulin-independentGLUT1, which is the predominant glucose transporter in bo-vine glycolytic muscle,29 may also compensate for the reducedGLUT4 expression in this muscle type. Thus, further studies arenecessary to examine the roles of GLUT1 and oxidative mus-cles in early lactation.

The deficit in ATP production through downregulation ofaconitase and MDH in the TCA cycle seems to be encounteredby (1) an increased expression of CK and ATP synthase in earlylactation and (2) a decreased expression of adenylate kinase1 (AK1; cytosolic myokinase) from calving to the second week oflactation. As a result, these metabolic adaptations likely contri-bute to ATP homeostasis and support muscle energy metabo-lism. An increased rate of lactate production through glycolysis aswell as increased expression of CK has also been observed in ratskeletal muscle during lactation.30

Muscle Fat MetabolismIt has been shown that cumulative feed intake increased in rats

after suppression of fatty acid oxidation inmuscle,16 suggesting a rolefor muscle fatty acid oxidation in the control of feed intake. Xiaoet al., (2004) have shown that skeletal muscle mRNA expression ofmolecules involved in fatty acid uptake and β-oxidation is dec-reased in lactating rats30 suggesting that reduced fatty acid oxidationmay contribute to favor feed intake during times of increased energyrequirement for milk production. However, in the present study wecould not identify enzymes directly involved in β-oxidation butfound ETF, which mainly participates in the oxidation of fatty acids,increases from the second to the fourth lactationweek. Furthermore,expression of RALDH1 which produces retinoic acid � a ligandpromoting peroxisome proliferator-activated receptor (PPAR)-regu-lated fatty acid metabolism � is also highest in the second week oflactation. These upregulations can be observed at the same timewhen plasma NEFA peaks and muscle fat content is lowest, sug-gesting a stimulated breakdown of triglycerides and increased fattyacid oxidation in the muscle during early lactation. Interestingly,elevated PPARR expression in the muscle coincides with reducedtriglyceride synthesis in the rat31 and pig32 muscle. Such a metabolicsituation would—in accordance to the study of Friedman et al.(1999)16—prevent a sufficient increase in postparturient feed intake.However, further studies are needed to resolve fatty acidmetabolismin the cow’s muscle during early lactation.

Muscle Protein Metabolism and GrowthThe continuous breakdown of muscle protein within the first

four weeks p.p. may be due to reduced protein synthesis orincreased proteolysis. In regard to reduced protein synthesis, weobserved reduced abundance of Dj-1 protein in early lactation,suggesting reduced cell growth promoting activity.33 Also, upre-gulation of PEBP1, a Raf kinase inhibitor, might suppress celldevelopment and growth. Second, we found reduced expressionof heat shock protein beta-1 (HSPB1) and R-crystallin. Bothproteins are involved in stabilization of myofibrillar protein, pro-

tection of actin, myosin and other cytoskeletons from degra-dation.34�36 Therefore, lowered HSPB1 and R-crystallin ex-pression around parturition suggests an increased proteasomaldegradation of these cytoskeletal proteins in particular, albeitHSPB1 and R-crystallin may also protect muscle during exerciseand inflammatory insults.37 As a third indicator of reducedmuscle protein in early lactation, we observed upregulation ofbridging integrator protein 1 (Bin1) which may activate caspase-independent apoptotic processes.38 Hence, the reduced totalmuscle protein content p.p. is likely attributed to the activation ofthe proteasome system, apoptotic processes and reduced pro-tein synthesis. Our findings confirm earlier studies that reportactivated components of the proteasomal machinery in earlylactation.6 Supplementing propylene glycol or dietary proteinmay influence the expression of the muscle proteolytic system inearly lactation;6,39 however, the peripartal changing nitrogenbalance occurs independently of the progression of age as studiedin 2 and 3 years old heifers.40Similar to the effects observed incows, early lactating sows show also robust expression of severalelements of the muscle ubiquitin proteasome, supporting proteinexport to the mammary gland and high growth rates in their up to12 offspring.41

Cytoskeletal and Structural Muscle ProteinsIn association with troponin, tropomyosin, R-actin and myo-

sin-1 form the contractile part of the skeletal muscle accountingfor 80% of the sarcomere or myofibrils. The lower abundant pro-teins vimentin and myotilin constitute the Z-disk. While R-actinand myosin seem to be early subjects for degradation becausetheir expression is lowest at parturition, vimentin shows its lowestabundance at 2 weeks p.p., and troponin at 4 weeks p.p. Tro-ponin, R-actin, vimentin, myosin but also myotilin are particu-larly rich in Glu, BCAA, Ala, Asp, and Lys which are all reducedby the fourth week p.p. (see above). However, the close associa-tion between cytoskeletal proteins and diverse chaperonesrevealed in the proteome analysis suggests not only a role formuscle plasticity involving contraction and distention34 but alsoin metabolic adaptation to increased energy demands duringlactation by providing AA. The PDZ and LIM domain protein 3(PDLIM3 or ALP) belongs to a family of adapter proteins thatbinds toR-actinin-2 at the Z lines of skeletal muscle. Although thefunction of PDLIM3 is not entirely resolved, a recent studysuggests a role for protein kinase C-mediated signaling via its LIMdomains, to R-actinin-2 through its PDZ domain.42 Thus, theobserved PDLIM3 peak at 2 weeks p.p. suggests the involvementof PDLIM3 in signaling for cytoskeletal degradation.

Transport ProteinsThe plasma transport protein albumin is known to transport

fatty acids and its increased abundance particularly after thesecond week of lactation may indicate an increasing requirementof FFA for replenishing intramuscular fat depots. Elevated levelsof myoglobin in the first 2 weeks p.p. refer to an increaseddemand of oxygen which is presumably needed for mitochon-drial ATP synthesis to compensate diminished ATP productionvia regulation of the TCA cycle (see above).

Neural PathwayPEBP1 has not only a function in cell growth (see above) but

also serves as precursor of the hippocampal cholinergic neuro-stimulatory peptide (HCNP) which may increase the productionof choline acetyltransferase in presynaptic cholinergic neurons.Cholinergic hypofunction is associated with reduced food intake43



while activation of hindbrain cholinergic neurons increased feedintake.44 These observations may provide a link between elevatedmuscle PEBP1 found in the present study and increased feedintake which is likely mediated via the nerval pathway.

’CONCLUSION

In summary, adaptations to the onset of lactation in muscle arecharacterized by an imbalanced plasma amino acid pattern,mobilization of protein, glycogen and fat reserves as well as byprotein expression changes pointing to an increase of cytoskeletalprotein and fat degradation, increased glycolysis and ATP pro-duction but a decrease in glycogenesis and TCA cycle activity.Considering our results and data from literature we propose amodel (Figure 7) in which the muscle’s metabolic (catabolic)state in early lactation supports the substrate supply for hepaticgluconeogenesis and milk production, and provide signals pre-sumably involved to modulate feed intake.

’ASSOCIATED CONTENT

bS Supporting InformationSupplemental Figure 1. Representative Colloidal Coomassie

stained 2-DGE of an individual muscle biopsy obtained twoweeks after parturition. Proteins were horizontally separated onan IPG gel strip (pH 3�10) and vertically on a 12.5% SDS PAGEgel (20 � 20 � 0.1 cm). Further characterization of spots issummarized in Supplemental Table 1. Supplemental Table 1.Muscle proteins identified by MALDI-TOF, MALDI-TOF/TOF and subsequent database search. Spot numbers refer tothose proteins shown in Figure 5 as clusters. This material isavailable free of charge via the Internet at http://pubs.acs.org.

’AUTHOR INFORMATION

Corresponding Author*Dr. Bj€orn Kuhla, Leibniz Institute for Farm Animal Biology(FBN), Research Unit Nutritional Physiology “Oskar Kellner”,Wilhelm-Stahl-Allee 2, 18196 Dummerstorf, Germany. Phone:+49-38208-68695. Fax: +49-38208-68652. E-mail: [email protected].

’ACKNOWLEDGMENT

We thankM. Althaus, K. Karpati, I. Br€uning, C. Fiedler, and thestaff at the FBN ‘Tiertechnikum’ for assistance with animal careand sample collection to performbiochemical analyses and proteinexpression studies. This study was supported by the core budget ofthe Leibniz Institute for Farm Animal Biology (FBN), Germany.

’ABBREVIATIONS

TMR, total mixed ration; NEL, net energy for lactation; a.p.,ante partum; p.p., postpartum.

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