Involvement of Skeletal Muscle Protein, Glycogen, and Fat Metabolism

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Involvement of Skeletal Muscle Protein, Glycogen, and Fat Metabolism in the Adaptation on Early Lactation of Dairy Cows Bj€orn Kuhla,*,† Gerd N€urnberg,‡ Dirk Albrecht,§ Solvig G€ors,† Harald M. Hammon,† and Cornelia 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 ABSTRACT: During early lactation, high-yielding dairy cows cannot consume enough feed to meet nutrient requirements. As a consequence, animals drop into negative energy balance and mobilize body reserves including muscle protein and glycogen for milk production, direct oxidation, and hepatic gluconeogenesis. To examine which muscle metabolic processes contribute to the adaptation during early lactation, six German Holstein cows were blood sampled and muscle biopsied throughout the periparturient period. From pregnancy to lactation, the free plasma amino acid pattern imbalanced and plasma glucose decreased. Several muscle amino acids, as well as total muscle protein, fat, and glycogen, and the expression of glucose transporter-4 were reduced within the first 4 weeks of lactation. The 2-DE and MALDI-TOF-MS analysis identified 43 differentially expressed muscle protein spots throughout the periparturient period. In early lactation, expression of cytoskeletal proteins and enzymes involved in glycogen synthesis and in the TCA cycle was decreased, whereas proteins related to glycolysis, fatty acid degradation, lactate, and ATP production were increased. On the basis of these results, we propose a model in which the muscle breakdown in early lactation provides substrates for milk production by 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

’ INTRODUCTION In the last days of gestation, dairy cows begin to reduce their feed intake until parturition and only slowly increase feed intake to reach a maximum by the sixth week postpartum. Thus, highyielding dairy cows ingest less nutrients and energy than they require for meeting energy demands of milk production during early lactation.1 As a consequence, animals experience negative energy balance in which they mobilize their fat, glycogen, and protein reserves. The release of body reserves particular that of fatty acids but also amino acids into circulation has been suggested to enable a high level of milk production but also to prevent sufficient feed intake in the early postparturient period.1 The mobilization of body protein in early lactation may amount to more than 20 kg in dairy cows.24 Much of this mobilized protein appears to be derived from peripheral tissues, primarily skeletal muscle5 and, to a lesser extent, skin, through suppression of tissue protein synthesis, and increased proteolysis.6,7 The released amino acids are intensively used for milk protein synthesis, direct oxidation, or gluconeogenesis but presumably to a different extent. As a result, this might cause an imbalanced r 2011 American Chemical Society

amino acid pattern in the circulation including the enrichment of certain amino acids.5,8,9 But although numerous amino acids exert highest satiating effects when compared with other macronutrients,10 the role of amino acids released from muscle as well as muscle metabolic processes potentially involved in regulating energy balance and feed intake during early lactation is still unknown. Within the circuits controlling feed intake, two major afferent pathways integrate protein and amino acid signals at the brain level: the indirect nervous-mediated and the direct blood pathway.10 Within the latter, the muscle as the predominant site of protein storage may undergo proteolysis around parturition and thereby releases creatinine, 3-methyl histidine,11,12 and other amino acids into circulation.5 Only recently has it been shown that elevated concentrations of leucine for example influence nutrient derived signaling in the central nervous system and reduces food intake.13 In addition, excess of specific amino acids, including Received: May 9, 2011 Published: July 21, 2011 4252

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

Journal of Proteome Research Met, Trp, and His, or increase in blood NH3 can exert toxic effects associated with aversive anorexic responses.10,14 Moreover, the muscle may release various myokines such as interleukin-6, interleukin-15, leukemia inhibitory factor (LIF), or brain-derived neurotrophic factor (BNDF) into the bloodstream to modulate energy metabolism and food intake via the direct pathway.15 The nervous-mediated pathway is also involved in the regulation of feed intake. For example, while inhibition of hepatic fatty acid oxidation alone does not influence feeding behavior, additional suppression of fatty acid oxidation in muscle stimulates food intake,16 suggesting the involvement of muscle metabolism in the control of feed intake. Also, ablation of nerves innervating skeletal muscles blocked the ability of intrahypothalamic injected leptin to stimulate fatty acid oxidation in the muscle.17 This effect is mediated via the R-adrenergic pathway resulting in the activation of muscle 50 -AMP-activated kinase (AMPK)—a key regulator of myocyte energy status.17 However, it is largely unknown how muscle proteolysis affects circulating amino acid concentrations and which metabolic pathways are activated or deactivated throughout the periparturient period. In order to identify these signals and metabolic events, we performed muscle amino acid profiling and proteome analysis to provide information on both amino acid release (direct pathway) as well as muscle metabolism (indirect pathway).

’ METHODS Animals and Biopsies

Six German Holstein cows in first and second lactation were ceased to be milked 9 weeks before expected calving (ages at that time: mean ( SD = 44 ( 5 month) and were fed a dry-off ration based on grass silage. With the beginning of the close-up period starting from 30 days before expected parturition and during lactation cows received ad libitum a total mixed ration (TMR) twice daily (4.30 a.m. and 11.30 a.m.). TMR consisted of corn and grass silage, hay and concentrate (6.4 MJ net energy lactation/ kg dry matter (NEL/kg DM) for the last 30 days of gestation (close-up period) and 7.0 MJ NEL/kg DM for lactation. NEL was calculated according to the German Society of Nutrition Physiology.18 Feed intake was recorded daily. During lactation, cows were milked twice daily (4.00 a.m. and 3.30 p.m.). Energy corrected milk yield increased to 50.7 kg/d by the fourth week postpartum. The semitendinosus muscles were biopsied after the morning milking and feeding, alternating left and right at days 24, +1, +14 and +28 (3, 0, +2, +4 weeks) relative to their second/3rd parturition using a custom-made biopsy shooting apparatus. Due to antibiotic therapy, one cow was not biospied at +1 day after parturition. The treatment was in accordance with the guidelines for the use of animals as experimental subjects of the State Government in Mecklenburg-West Pommerania (Registration No. LALLF M-V/TSD/7221.32.1019/08). Sample Preparation for 2-DE Analyses

The biopsy (∼0.5 mm 3 cm; approximately 500 mg) was liberated from skin and subcutaneous fat and muscle tissue was shock-frozen in liquid N2. Frozen muscle tissue was crushed to a fine 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 (all from Amersham Biosciences, Uppsala, Sweden). After centrifugation

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(11 000 g, 4 °C, 20 min), the protein concentration in the supernatant was measured according to the Bradford method using BSA as standard. 2-D Electrophoresis

Individual muscle samples (n = 23) were run in technical duplets yielding 46 gels in total. A sample of 500 μg protein was added to 320 μL rehydration buffer (8 M urea, 2% CHAPS, 0.8% IPG-buffer, 18 mM DTT and a trace of bromophenol blue), mixed and loaded to 18 cm IPG (pH 310) (Amersham Biosciences, Uppsala Sweden). After rehydration and IEF, the IPG was equilibrated in buffer containing 50 mM Tris (pH 8.8), 30% glycerol, 6 M urea, 2% SDS, 1% DTT and than in the same buffer without DTT but 2.5% iodoacetamide, each for 15 min. IPGs were transferred to 12.5% SDS PAGE gels (20 20 0.1 cm) and embedded in low melting agarose. The gels were stained overnight in colloidal Rotiblue (Roth, Karlsruhe, Germany), destained 3-times in 15% methanol and 5% acetic acid and 1 in distilled water. Image Analysis

Gels were scanned using an Epson Perfection 1250 scanner and saved as tiff format (8-bit gray scale). The 2-DE image analysis was carried out on a computer using Delta2D software version 4.0 (DECODON, Greifswald, Germany; http://www. decodon.com). Gels derived from one animal were warped according to the “all to one” warping strategy. A fusion image was created from all warped images containing all spots from all gels. After automatic spot detection on the fusion image, spot boundaries were transferred to the original images and there quantified using the gray value of each spot to obtain the spot volume. Each spot volume was normalized to the total spot volume of each gel image (=100%) yielding the normalized spot volume in % which was further used for statistical analysis (see below). Mass Spectrometry

Protein identification was performed by the method compiled previously.19 Briefly, protein spots were punched out using an Ettan spot cutter (Amersham) with a 2 mm picker head. Spots were transferred into 96 well micro titer plates, tryptic digested and subsequently spotted on a MALDI-target. The molecular masses of tryptic digests were measured on a 4800 MALDI TOF/TOF Analyzer (Applied Biosystems). The spectra were recorded in a mass range from 900 to 3700 Da with a focus on 2000 Da. For one main spectrum, 30 subspectra with 60 shots per subspectrum were accumulated. When the autolytical fragments of trypsin with (M + H)+ m/z at 1045.556 and 2211.104 reached a signal-to-noise ratio (S/N) of at least 20, an internal calibration was automatically performed as two-point-calibration. The standard mass deviation was less than 0.15 Da. After calibration the peak lists were created by using the “peak to mascot” script of the 4000 Series Explorer Software (V3.5). Selected settings were: mass range from 900 to 3800 Da, peak density of 15 peaks per 200 Da, minimal area of 100 and maximal 60 peaks per spot. The peak list was created for an S/N ratio of 10. To confirm the results obtained by MALDI-TOF-MS, MALDI-TOF-TOF analysis was on the 4800 MALDI TOF/TOF Analyzer (Applied Biosystems). The three strongest peaks of the TOF-spectra were selected automatically and measured. For one main spectrum 25 subspectra with 125 shots per subspectrum were accumulated using a random search pattern. The internal calibration was automatically performed as one-point-calibration with (M+H)+ m/z at 175.119 or with lysine (M + H)+ m/z at 4253

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

Journal of Proteome Research

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Table 1. Complete List of Differentially Expressed Proteins (ANOVA p < 0.05; Tukey test p < 0.1) and Affiliation to Cluster Analysis 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 16

R-actin R-actin

56204817 56204817

1 1

3 vs 0; 3 vs 0; 2 vs 4 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

63 186

Myotilin (MYOT) troponin T, beta isoform (TNNT)

116734833 259912

8 4

3 vs 4; 0 vs 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

51

glyceraldehyde-3-phosphate dehydrogenase

163866419

7

0 vs 4

174

glyceraldehyde-3-phosphate dehydrogenase

77404273

8

3 vs 4; 0 vs 4

124 173

glyceraldehyde-3-phosphate dehydrogenase glyceraldehyde-3-phosphate dehydrogenase

40889050 77404273

1 4

0 vs 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

Glycolysis and Glycogenesis

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

119920080

6

3 vs 2

19

UTP-glucose-1-phosphate uridylyltransferase (UGP1)

41386780

3

0 vs 2

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


4838363

10

118


4838363

6

3 vs 2; 0 vs 2; 2 vs 4

45 150

ATP synthase, subunit alpha (ATP5A) adenylate kinase 1 (AK1)

27807237 61888850

7 3

3 vs 0 0 vs 2

23

retinal dehydrogenase 1 (RALDH 1)

160332357

5

0 vs 2

38

electron transfer flavoprotein, subunit alpha (ETFA)

115496196

10

2 vs 4

dehydrogenase, A chain (LDH-A)

TCA cycle and ATP homeostasis

2 vs 4

Fatty acid oxidation

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

73

heat shock protein beta-1 (HSPB1)

71037405

8

2 vs 4 3 vs 4

Binding and transport

a

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 4

Spot number refers to Supplementary Figure 1, Supporting Information. 4254



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Glucose, Urea and Amino Acid Analyses

Figure 1. Average daily DM intake of dairy cows ranging between week 4 a.p. and week 5 p.p. The value at week “0” represents the DM intake at the day of calving. Data are presented as mean ( SEM. The gray arrows illustrate the day of muscle biopsy sampling. DM intake significantly differed over time at P < 0.001 (ANOVA). When compared to the day of calving, feed intake between week +2 and +5 relative to parturition significantly differed (*) at P < 0.05 (Tukey-test).

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 ratio of 7. Selected settings were: mass range from 60 to precursor 20 Da, peak density of 15 peaks per 200 Da, minimal area of 100 and maximal 65 peaks per precursor. For the identification of proteins, database search with PMF of the analyte was performed against the databases NCBInr (National Center for Biotechnology Information, http://www.ncbi.nlm.nih. gov/) and Swiss-Prot (http://us.expasy.org/sprot/) using the Mascot search engine version 2.1 (Matrix Science Ltd., London, U.K.). Search parameters were taxonomy: “all entries”; variable modifications: “carbamidomethyl (C)” and “oxidation (M)”; peptide tolerance (50 ppm; peptide charge “1+”; MS/MS tolerance “0.5 Da”; “monoisotopic”. Determination of Muscle Protein, Fat and Glycogen Content

Tissue powder (50 mg) was dried in a muffle type furnace at 105 °C for 3 h to determine content of dry matter (DM). Dried samples were analyzed for nitrogen and carbon content at an element CNS 2000 analyzer (LECO Instrumente GmbH, M€onchengladbach, Germany). Fat and protein content were calculated according to the method described previously.20 For the analysis of glycogen, 25 mg wet tissue was applied to an enzyme-based starch kit (#10207748035; Boehringer Mannheim, Germany) according to the manufacture’s introduction. Western Blot Analysis

Due to the limited amount of biopsy material powdered muscle tissue (20 mg) from only three animals was extracted in 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 at 13.000 rpm for 10 min at 4 °C. Protein extracts were treated with Laemmli buffer, boiled for 5 min, loaded on a 12% SDS gel, and electrotransferred to nitrocellulose. Blots were reversibly stained with Ponceau S visualizing total proteins, washed and incubated with rabbit anti GLUT4 IgG (each 1:750; Biotrend, Germany) for 16 h at 4 °C. After 5 washing steps in TBST (TBS containing 0.05% Tween 20), horseradish peroxidase-labeled anti rabbit antibody (1:10 000; Santa Cruz, Santa Cruz, CA) was applied for 2 h at room temperature. After washing, blots were developed on hyperfilmes using Enhanced Chemiluminescence (ECL) reaction. Hyperfilms and Ponceau stained blots were scanned and digital images were analyzed by ImageJ software.

Blood was collected from the jugular vein at day 24, 7, +1, +7, +15, +28 (3, 1, 0, +1, +2, +4 weeks) relative to parturition in sodium EDTA tubes (Sarstedt AG & CO, N€umbrecht, Germany). Blood samples were centrifuged for 20 min at 4 °C and 2000 g and the plasma frozen at 80 °C for later analyses. Plasma glucose and urea were analyzed by kits LT GL 0251 (for glucose) and LT UR 0010 (for urea) purchased from Labor and Technik Lehmann (Berlin, Germany). All measurements were performed by an automatic analyzer (Cobas Mira Plus; Roche, Basel, Switzerland). For amino acids analyses, plasma samples were diluted with water (1:10) and free amino acids (FAA) were analyzed by HPLC equipped with a fluorescence detector (Series 1200; Agilent Technologies, Waldbronn, Germany) as described previously.19 Therein, precolumn derivatization was performed with orthophthalaldehyde (OPA)/3-mercaptopropionic acid (MCPA) for primary and 9-fluorenylmethoxycarbonyl chloride (FMOC) for secondary amino acids using MCPA as reducing agent and iodoacetic acid to block sulfhydryl groups. Standard mixtures of amino acids (completed with asparagine and glutamine) allowed assignment of retention times and quantification. For the analysis of muscle amino acids (free and proteinogenic), 5 mg of powdered tissue was treated with 2 mL 6 N HCl and incubated at 110 °C for 24 h. The hydrolysate was dried at 60 °C under N2 atmosphere, dissolved in 4 mL water, and subjected to HPLC as described above. Statistics

For statistical evaluation of feed intake, intermediary plasma metabolites, and muscle amino acids, glycogen and total fat, as well as protein expression pattern, an one-way repeated measures ANOVA analysis was calculated with repeated factor time (e.g., for biopsies: 3, 0, +2, +4 weeks relative to parturition; see above). As posthoc test, we used Tukey’s multiple comparison test for pairwise differences between time points to ensure multiple comparison adjustment for the p-values. For evaluation of the protein expression time course, we only considered those protein spots from the repeated measure ANOVA analysis with at least one significant expression difference between two time points (Tukey adjusted p-value < 0.10; see Table 1). Following the aim to group proteins with a similar expression—time course, we rescaled the normalized spot volume values obtained from Image analysis (see above) at time point 0, +2, +4 (postpartum) relative to time point 3 (ante partum). To this end, the expression value at time point 3 was set equal to 1 and the expression values at time point 0, +2, +4 were divided by the expression value at time point 3. By this way, one gets more comparable, relative expression courses independently of the gray value-based expression scale. With these relative expression values, we conducted a cluster analysis using proc VARCLUS of SAS (2009).21 After clustering, means of the relative expression values of those spots forming one cluster were calculated (see Figure 5).

’ RESULTS DM Intake and Plasma Amino Acid Changes in Early Lactation

DM intake continuously declined starting 4 weeks before parturition until calving to 81% (P < 0.01) and increased until the 4255



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

fifth week after parturition gaining 163% in comparison to the intake at calving (P < 0.001; ANOVA) (Figure 1). According to the course of DM intake, plasma concentrations of free essential amino acids (EAA) - except of His and Met show a nadir at the first day after calving (Figure 2). His, Met and the nonessential amino acids (NEAA) Asp, Gln, Glu, Cys, Tyr, and Orn as well as plasma glucose continuously decrease until calving and remain lower in early lactation than days a.p. (P < 0.05, ANOVA). In contrast, concentrations of Gly, Tau, and R-aminobutyric acid (R-ABA) continuously increase starting either one week before or at calving until the fourth week after p.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 of lactation but only the latter two reach significance level (P < 0.05, ANOVA). Muscle Amino Acid, Glycogen and Fat in Early Lactation

To 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 the amino acid profile in the muscle. In the fourth week p.p., muscle EAA, Gln/Glu, Asp/Asn, Tyr, and Ser are reduced to ∼86% when compared to the third week a.p. (Figure 3). Whereas muscle Pro, His, Tau, β-Ala, and Me-His levels did not change over time (P > 0.1), Gly levels (P < 0.07) are lowest at parturition (Figure 3). Likewise, muscle glycogen and total fat content reach a nadir at 2 weeks p.p. whereas total muscle protein tends to decrease after parturition (Figure 4). Metabolic Changes in Early Lactation

In order to elucidate cellular and metabolic processes underlying the mobilization of energy reserves stored in muscle tissue, we performed a 2D-GE based proteome analysis. Out of 601 spots detected at the gel image, we picked all clearly separated and visually recognizable spots (n = 250) and among them identified 181 spots by MALDI-TOF mass spectrometry (Supplementary Figure 1, Supplementary Table 1, Supporting Information). Among them, we found 75 spots of cytoskeletal origin, 14 spots with binding and transport properties, 20 spots 4256



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Figure 3. Amino acid composition of muscle hydrolysates during the periparturient period. In the first row essential amino acids, in the second and third row 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 P e 0.05 between the ante partum and postpartum period whereas # shows differences with P < 0.1 (Tukey-test).

referring to protein and amino acid metabolism, 48 spots representing enzymes involved in glycolysis and TCA cycle, 15 spots referring to pH and ATP homeostasis, and 9 spots with miscellaneous function including regulators of fat metabolism (Supplemental Table 1, Supporting Information). Statistical analyses revealed that 43 of the identified 181 spots are differentially regulated throughout the periparturient period (Table 1). These 43 spots were assigned to 10 clusters according to a similar expression-time course (Figure 5). In early lactation, there is increased expression of glycolytic enzymes such as fructose bisphosphate aldolase A, GAPDH, triosephosphate isomerase, enolase, pyruvate kinase and lactate dehydrogenase (123198%) as compared to the third week a.p. Conversely, we found decreased glycogenesis as indicated by the lowest expression of UTP-glucose-1-phosphate uridylyltransferase (∼55%) 2 weeks p.p. Furthermore, expression of TCA cycle enzymes (aconitase 2, malate dehydrogenase; MDH) is also lowered (5474%) 4 weeks p.p. whereas expression of enzymes regulating ATP homeostasis such as creatine kinase (CK) and ATP synthase are upregulated (125185%) as compared to late pregnancy. Two proteins closely associated with fatty acid degradation namely electron transfer flavoprotein (ETF) and retinal dehydrogenase 1 (RALDH1) are also increasingly expressed (196 and 162%, repectively) in early lactation. Furthermore, we observed coordinated downregulation of proteins involved in protein synthesis and stabilization (Dj-1 protein, heat shock protein beta-1 (HSBP1) and its binding partner R-crystallin as well as upregulation of the cell growth

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

suppressor phosphatidylethanolamine-binding protein 1 (PEBP1) in early lactation. During this period, a number of cytoskeletal and structural proteins such as R-actin, myosin, myotilin, troponin, vimentin, and R-crystallin were also decreased (6441%). The abundance of the transport protein myoglobin peaked in the second week p.p. whereas that of serum albumin was highest 4 weeks p.p. 4257



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

tissue. GLUT4 is insulin-regulated and due to the insulin deficiency expected to be altered in early lactation. GLUT4 Western blot analysis was performed—similar to the proteome image analysis—by normalization to the total protein load stained with Ponceau (∼20250 kDa), because cytoskeletal proteins as well as GAPDH potentially serving as housekeeping genes were regulated (see above). We found that relative expression of GLUT4 is reduced to ∼60% in the fourth week p.p. as compared to the level around parturition (Figure 6).

’ DISCUSSION Plasma AA

Figure 6. Western Blot analysis of muscle GLUT4 in three periparturient cows. Tissue extracts underwent electrophoresis and blotting. Blots were stained by Ponceau and subsequent immunoblotted for GLUT4. For densitometrical analysis GLUT4 immunoreactivity was normalized to the total protein content of the blot as stained by Ponceau. Foldchange of expression relative to week 3 was calculated and data are shown as mean + SEM. Pair-wise comparisons are indicated by * (Tukey test; P < 0.05).

GLUT4 Expression in Early Lactation

To examine whether increased muscle glycolytic processes described above are supplied by glycogen degradation or increased glucose uptake from the circulation, we examined the expression of the glucose transporter 4 (GLUT4) in muscle

Because of the continuous outflow of microbial and undegraded dietary protein from the rumen, absorption and peripheral plasma concentrations in dairy cows are relatively unchanged during the day and thus unaffected by variation in time after the last milking or meal intake.22 During late gestation, however, fetal growth, lactogenesis and during early lactation milk protein synthesis but also gluconeogenesis require enormous amounts of certain AA that are withdrawn from the circulation resulting in an imbalanced AA pattern in the blood. This can be seen in most plasma FAA falling immediately after parturition. Another reason for decreasing plasma AA may be the reduced DM intake around parturition that reduces the supply from the intestinal tract and thus additionally contributes to an imbalanced plasma AA pattern. However, increased DM intake after the second week of lactation supports the recovery of the concentration of most of the FAA felt around parturition. Our results are in agreement with earlier studies,5,8,9 which reported FAA concentrations over a longer postparturient period. The plasma concentrations of total NEAA, primarily those of plasma Tau, Ser, Asn, Gly, Hy-Pro, R-ABA, and Me-His increase after parturition and remain elevated as compared to the a.p. period, suggesting that body protein is degraded. This degradation is directed to counteract the circulating AA imbalance which 4258



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Figure 7. Schematic representation of the interaction between protein expression patterns in muscle and physiological events in liver and mammary gland during early lactation. We propose that (1) released amino acids primarily from cytoskeletal degradation are used by the liver as glucoplastic precursors or by the mammary gland for milk protein synthesis and free AA. (2) Muscle glycogen is used in the glycolysis pathway yielding pyruvate which is converted under anaerobic conditions toward lactate. Lactate serves again as precursor for hepatic gluconeogenesis. Because of the reduced GLUT4 expression in muscle and the high glucose demand by the mammary gland, hypoglycaemia prevails resulting in a decoupled Cori cycle in early lactation. Since pyruvate is not converted to AcCoA, the activity of the TCA cycle is diminished and as a compensatory mechanism ATP is produced 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.

in turn is driven by withdrawal of amino acids for gluconeogenesis and milk protein synthesis. For example, when the contribution of Ala increase to as much as 5% for hepatic gluconeogenesis in early lactation,23 plasma Ala does not fall suggesting that whole body protein degradation copes with the increased demand of glucogenic precursors. Other amino acids mobilized from body protein in early lactation are mainly directed to milk protein synthesis24 leading to a deficiency of certain AA in the circulation. On the other hand, mobilized AA that are not used for synthetic processes and exceed oxidative capacity may accumulate in plasma and thus contribute to AA imbalance. For example, Me-His, which occurs only in actin and myosin accumulates in early lactation and has been suggested to predict milk protein yield.11 Interestingly, R-ABA has been shown to be released from forearm muscle tissue after short-term starvation in man25 but it has not been analyzed in altered metabolic conditions of cows so far. Here we describe for the first time that R-ABA is 2.5-times elevated in early lactation but not after feed restriction for 60 h;26 however, increased R-ABA may also be generated by accelerated conversion of cystathionine in the liver.27 Cystathionine is produced from Met metabolism and interestingly, Met concentrations declined after calving. Also, the ratio of R-ABA/ cystathionine may predict fatty liver disease in humans27,28 and dairy cows similarly show signs of increased liver fat content in early lactation.1

earlier result describing increasing muscle FAA concentrations from late pregnancy to lactation.5 Rather our findings suggest that degradation of muscle proteins contributes to these accumulated FAA in muscle during early lactation. Just recently, various muscle protein degradation systems including the caspase, the Ca2+dependent, and the ATP-dependent ubiquitin-mediated system were found to be activated in early lactation and thus likely account for the muscle protein breakdown6 (see also below). Muscle Ala levels fall only numerically by the fourth week of lactation suggesting only a minor role of nitrogen transport from the muscle to the liver within the glucose-alanine cycle. Also, because muscle Gly and Me-His levels are not reduced in the early postparturient period, it appears that increased plasma Gly and Me-His concentrations in early lactation are not of semitendinosus muscle origin. While muscle protein and amino acid losses continuously progress within the first weeks of lactation, muscle glycogen and fat storages are already exhausted immediately after parturition. This observation points to an early allocation of glucose and fatty acids and a latter allocation of AA either directly for milk production or hepatic anabolic processes in early lactation. In summary, skeletal muscle tissue contributes to the adaptation to early lactation by mobilizing tissue constitutes in the order of glycogen, fat and protein. Muscle Glycolysis, TCA Cycle, and ATP Homeostasis

Muscle AA

The amount of FAA in the muscle reflects the largest part of the FAA pool in the body and was previously analyzed in periparturient cows.5 However, changes in FAA in the intracellular muscle fluid do not necessarily reflect muscle tissue breakdown. Hence, we measured total (free and proteinogenic) AA in muscle biopsies before and after parturition. With the exception of Pro, His, Tau, Gly, β-Ala, and Me-His, all muscle AA determined but also the total protein content decreased p.p. indicating a pronounced negative N balance. This finding is not in contrast to an

Muscle glycogen degradation likely results in the activation of the glycolysis pathway as indicated by peaking expression of five glycogenolytic and glycolytic enzymes and decreased expression of one glycogen synthesis enzyme within the first two weeks of lactation. Additionally, we observed highest expression of lactate dehydrogenase in the second week of lactation and continuously falling levels of TCA cycle enzymes by the fourth week of lactation, suggesting that (1) pyruvate is shunted toward lactate (rather than toward AcCoA) and (2) ATP production through the TCA cycle is decreased. Increased skeletal muscle lactate 4259


Journal of Proteome Research production presumably serves as substrate for hepatic gluconeogenesis. Its contribution may reach up to 1525% in early lactation.23 Considering the hypoglycaemic conditions, the prioritized glucose partitioning to the mammary gland, together with the reduced expression of muscle GLUT4, we conclude that during early lactation the Cori cycle involving glycolytic muscle is decoupled (Figure 7). However, since the expression of GLUT4 in bovine glycolytic muscle is lower than in more oxidative muscle,29 GLUT4 in oxidative muscle might compensate for the reduced GLUT4 expression in glycolytic muscle. Moreover, beside the insulin-regulated GLUT4, the insulin-independent GLUT1, which is the predominant glucose transporter in bovine glycolytic muscle,29 may also compensate for the reduced GLUT4 expression in this muscle type. Thus, further studies are necessary to examine the roles of GLUT1 and oxidative muscles in early lactation. The deficit in ATP production through downregulation of aconitase and MDH in the TCA cycle seems to be encountered by (1) an increased expression of CK and ATP synthase in early lactation and (2) a decreased expression of adenylate kinase 1 (AK1; cytosolic myokinase) from calving to the second week of lactation. As a result, these metabolic adaptations likely contribute to ATP homeostasis and support muscle energy metabolism. An increased rate of lactate production through glycolysis as well as increased expression of CK has also been observed in rat skeletal muscle during lactation.30 Muscle Fat Metabolism

It has been shown that cumulative feed intake increased in rats after suppression of fatty acid oxidation in muscle,16 suggesting a role for muscle fatty acid oxidation in the control of feed intake. Xiao et al., (2004) have shown that skeletal muscle mRNA expression of molecules involved in fatty acid uptake and β-oxidation is decreased in lactating rats30 suggesting that reduced fatty acid oxidation may contribute to favor feed intake during times of increased energy requirement for milk production. However, in the present study we could not identify enzymes directly involved in β-oxidation but found ETF, which mainly participates in the oxidation of fatty acids, increases from the second to the fourth lactation week. Furthermore, expression of RALDH1 which produces retinoic acid a ligand promoting peroxisome proliferator-activated receptor (PPAR)-regulated fatty acid metabolism is also highest in the second week of lactation. These upregulations can be observed at the same time when plasma NEFA peaks and muscle fat content is lowest, suggesting a stimulated breakdown of triglycerides and increased fatty acid oxidation in the muscle during early lactation. Interestingly, elevated PPARR expression in the muscle coincides with reduced triglyceride synthesis in the rat31 and pig32 muscle. Such a metabolic situation 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 acid metabolism in the cow’s muscle during early lactation. Muscle Protein Metabolism and Growth

The continuous breakdown of muscle protein within the first four weeks p.p. may be due to reduced protein synthesis or increased proteolysis. In regard to reduced protein synthesis, we observed reduced abundance of Dj-1 protein in early lactation, suggesting reduced cell growth promoting activity.33 Also, upregulation of PEBP1, a Raf kinase inhibitor, might suppress cell development and growth. Second, we found reduced expression of heat shock protein beta-1 (HSPB1) and R-crystallin. Both proteins are involved in stabilization of myofibrillar protein, pro-

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tection of actin, myosin and other cytoskeletons from degradation.3436 Therefore, lowered HSPB1 and R-crystallin expression around parturition suggests an increased proteasomal degradation of these cytoskeletal proteins in particular, albeit HSPB1 and R-crystallin may also protect muscle during exercise and inflammatory insults.37 As a third indicator of reduced muscle protein in early lactation, we observed upregulation of bridging integrator protein 1 (Bin1) which may activate caspaseindependent apoptotic processes.38 Hence, the reduced total muscle protein content p.p. is likely attributed to the activation of the proteasome system, apoptotic processes and reduced protein synthesis. Our findings confirm earlier studies that report activated components of the proteasomal machinery in early lactation.6 Supplementing propylene glycol or dietary protein may influence the expression of the muscle proteolytic system in early lactation;6,39 however, the peripartal changing nitrogen balance occurs independently of the progression of age as studied in 2 and 3 years old heifers.40Similar to the effects observed in cows, early lactating sows show also robust expression of several elements of the muscle ubiquitin proteasome, supporting protein export to the mammary gland and high growth rates in their up to 12 offspring.41 Cytoskeletal and Structural Muscle Proteins

In association with troponin, tropomyosin, R-actin and myosin-1 form the contractile part of the skeletal muscle accounting for 80% of the sarcomere or myofibrils. The lower abundant proteins vimentin and myotilin constitute the Z-disk. While R-actin and myosin seem to be early subjects for degradation because their expression is lowest at parturition, vimentin shows its lowest abundance at 2 weeks p.p., and troponin at 4 weeks p.p. Troponin, R-actin, vimentin, myosin but also myotilin are particularly rich in Glu, BCAA, Ala, Asp, and Lys which are all reduced by the fourth week p.p. (see above). However, the close association between cytoskeletal proteins and diverse chaperones revealed in the proteome analysis suggests not only a role for muscle plasticity involving contraction and distention34 but also in metabolic adaptation to increased energy demands during lactation by providing AA. The PDZ and LIM domain protein 3 (PDLIM3 or ALP) belongs to a family of adapter proteins that binds to R-actinin-2 at the Z lines of skeletal muscle. Although the function of PDLIM3 is not entirely resolved, a recent study suggests a role for protein kinase C-mediated signaling via its LIM domains, to R-actinin-2 through its PDZ domain.42 Thus, the observed PDLIM3 peak at 2 weeks p.p. suggests the involvement of PDLIM3 in signaling for cytoskeletal degradation. Transport Proteins

The plasma transport protein albumin is known to transport fatty acids and its increased abundance particularly after the second week of lactation may indicate an increasing requirement of FFA for replenishing intramuscular fat depots. Elevated levels of myoglobin in the first 2 weeks p.p. refer to an increased demand of oxygen which is presumably needed for mitochondrial ATP synthesis to compensate diminished ATP production via regulation of the TCA cycle (see above). Neural Pathway

PEBP1 has not only a function in cell growth (see above) but also serves as precursor of the hippocampal cholinergic neurostimulatory peptide (HCNP) which may increase the production of choline acetyltransferase in presynaptic cholinergic neurons. Cholinergic hypofunction is associated with reduced food intake43 4260


Journal of Proteome Research while activation of hindbrain cholinergic neurons increased feed intake.44 These observations may provide a link between elevated muscle PEBP1 found in the present study and increased feed intake which is likely mediated via the nerval pathway.

’ CONCLUSION In summary, adaptations to the onset of lactation in muscle are characterized by an imbalanced plasma amino acid pattern, mobilization of protein, glycogen and fat reserves as well as by protein expression changes pointing to an increase of cytoskeletal protein and fat degradation, increased glycolysis and ATP production but a decrease in glycogenesis and TCA cycle activity. Considering our results and data from literature we propose a model (Figure 7) in which the muscle’s metabolic (catabolic) state in early lactation supports the substrate supply for hepatic gluconeogenesis and milk production, and provide signals presumably involved to modulate feed intake. ’ ASSOCIATED CONTENT

bS

Supporting Information Supplemental Figure 1. Representative Colloidal Coomassie stained 2-DGE of an individual muscle biopsy obtained two weeks after parturition. Proteins were horizontally separated on an IPG gel strip (pH 310) and vertically on a 12.5% SDS PAGE gel (20 20 0.1 cm). Further characterization of spots is summarized in Supplemental Table 1. Supplemental Table 1. Muscle proteins identified by MALDI-TOF, MALDI-TOF/ TOF and subsequent database search. Spot numbers refer to those proteins shown in Figure 5 as clusters. This material is available 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: b.kuhla@ fbn-dummerstorf.de.

’ ACKNOWLEDGMENT We thank M. Althaus, K. Karpati, I. Br€uning, C. Fiedler, and the staff at the FBN ‘Tiertechnikum’ for assistance with animal care and sample collection to perform biochemical analyses and protein expression studies. This study was supported by the core budget of the 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. ’ REFERENCES (1) Ingvartsen, K. L.; Andersen, J. B. Integration of metabolism and intake regulation: a review focusing on periparturient animals. J. Dairy Sci. 2000, 83, 1573–1597. (2) Komaragiri, M. V.; Erdman, R. A. Factors affecting body tissue mobilization in early lactation dairy cows. 1. Effect of dietary protein on mobilization of body fat and protein. J. Dairy Sci. 1997, 80, 929–937.

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Involvement of Skeletal Muscle Protein, Glycogen, and Fat Metabolism

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