Hepatic Gene Expression of Apolipoprotein B100 During Early Lactation in Underfed, High Producing Dairy Cows1

Hepatic Gene Expression of Apolipoprotein B100 During Early Lactation in Underfed, High Producing Dairy Cows1 DOMINIQUE GRUFFAT,*,2 DENYS DURAND,* YVES CHILLIARD,† PETER WILLIAMS,‡ and DOMINIQUE BAUCHART* *Unite´ de Recherches Me´tabolismes Energe´tique et Lipidique, Laboratoire Croissance et Me´tabolismes des Herbivores, Institut National de Recherche Agronomique, Centre de Recherches de Clermont-Ferrand, Theix, 63122 St-Gene`s Champanelle, France †Laboratoire Sous-Nutrition des Ruminants, Institut National de Recherche Agronomique, Centre de Recherches de Clermont-Ferrand, Theix, 63122 St-Gene`s Champanelle, France ‡Rhoˆne-Poulenc Animal Nutrition, 42 Avenue A. Briand, BP 100, 92164 Antony Cedex, France

ABSTRACT The hepatic gene expression of apolipoprotein B, the major protein of very low density lipoproteins in plasma, was studied using 8 Holstein × Friesian cows during the first 12 wk of lactation. Cows were fattened during gestation and were underfed just after parturition to increase fat mobilization and subsequent hepatic steatosis. Intracellular concentrations of apolipoprotein B and apolipoprotein B mRNA and control parameters (albumin, total lipids, RNA, and proteins) were determined in liver samples obtained by biopsy from each cow on four occasions at 1, 2, 4, and 12 wk after calving. Results were compared with those obtained from 5 dry cows in late pregnancy and 4 dry nonpregnant cows. The hepatic concentration of apolipoprotein B was lower (approximately 25%) during wk 1, 2, and 4 after calving, a period of intense liver steatosis (44.2 to 95.7 mg of triglycerides/g of fresh tissue), than for nonsteatotic dry cows (pregnant or nonpregnant); hepatic concentrations were also lower than those during wk 12. In contrast, hepatic concentrations of mRNA coding for apolipoprotein B, total proteins, RNA, and albumin did not vary significantly during early lactation. These results suggested that synthesis of apolipoprotein B during early lactation is specifically

Received January 22, 1996. Accepted August 1, 1996. 1Part of the present work was presented at the 9th Annual Conference on Nutrition and Feeding of Herbivores organized by the French National Institute of Agronomical Research (ClermontFerrand, March 1994) and was published as an abstract (19). 2Reprint requests. 1997 J Dairy Sci 80:657–666

regulated at a posttranscriptional level by a decrease in the rate of translation, or by a higher rate of intracellular degradation of apolipoprotein B, or both. ( Key words: apolipoprotein B, gene expression, dairy cow, liver) Abbreviation key: apo = apolipoprotein (used with B), FA = fatty acids, LDL = low density lipoproteins, TG = triglyceride, TTBS = Tris-buffered saline containing 5 ml/L of Tween, VLDL = very low density lipoproteins. INTRODUCTION Fatty liver arises in response to various nutritional, hormonal, or toxic effects. Fatty liver has been reported as a physiological or pathological situation resulting from excessive natural or induced mobilization of fat (17, 20). During the 1st mo of lactation, high producing dairy cows can mobilize large amounts of fatty acids ( FA) from adipose tissue to compensate for negative energy balance caused by stress at calving, underfeeding, initiation of milk production, hormonal imbalance, or a combination of these factors (9, 21). During this period, plasma accumulation of NEFA provides large amounts of long-chain FA that can be taken up by the liver by mass action. Such conditions enhance triglyceride ( TG) synthesis and subsequent accumulation in hepatocytes (21), which can frequently alter the structure and functions of the liver (35). Extension of this phenomenon, generally associated with ketosis, is amplified by undernutrition ( 9 ) . Studies carried out on goat hepatocytes in culture ( 2 3 ) and on sheep liver ( 3 2 ) clearly indicated that secretion of TG by the liver of ruminants as components of very low

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density lipoproteins ( VLDL) is low compared with that occurring in primates and rodents. Various studies (3, 4, 31) have suggested that, in early lactation, hepatic synthesis of apolipoprotein ( apo) B is impeded in cows with fatty liver. Apolipoprotein B represents the major protein of the VLDL particles and lipoproteins that results from their catabolism by the lipolytic cascade [intermediate density lipoproteins and low density lipoproteins ( LDL) ] (3, 8). This apolipoprotein is a large protein that contains several different domains involved in lipid binding and in the tissue internalization of particles containing apo B by receptor-mediated endocytosis (LDL receptor) ( 8 ) . Synthesis and secretion of hepatic apo B are probably regulated by nutritional and hormonal factors ( 1 ) , but the specific mechanisms remain largely unknown. The results obtained for rodents and humans indicated that hepatic concentrations of apo B mRNA were not modified by acute stimuli. These results were consistent with the observation that, in the human hepatoma cell line HepG2, the half-life of apo B mRNA is relatively long (16 h ) (33). Thus, secretion of apo B apparently is not modulated transcriptionally, but rather translationally or posttranslationally. Thus, among different possible mechanisms operating in mammals, intracellular translation of apo B mRNA and degradation of apo B are more likely to be the key regulatory mechanisms controlling the acute regulation of apo B production by the liver ( 1 ) . The objectives of the work were 1 ) to examine changes in gene expression of apo B in hepatocytes of fat and underfed dairy cows during the first 3 mo of lactation and of dry pregnant or nonpregnant cows and 2 ) to relate changes in gene expression with hepatic characteristics in lipid and protein metabolism throughout the dry period, gestation, and early lactation. MATERIALS AND METHODS Cows and Diets A total of 17 Holstein cows were used in this study. One group consisted of 4 multiparous nonpregnant dry cows (665 ± 10 kg of BW; 6.6 ± 3.2 yr old) and 5 multiparous pregnant dry cows (717 ± 18 kg of BW; 4.4 ± 1.1 yr old) in late gestation (21 ± 9 d before calving) that were kept on pasture until the day of sampling. Pregnant cows were provided a diet supplemented with corn silage for ad libitum intake and concentrate ( 4 kg/d per cow). A second group conJournal of Dairy Science Vol. 80, No. 4, 1997

sisted of 8 multiparous cows (4.6 ± 1.4 yr old) that were fattened before calving [target body score at 1 mo before calving > 4 on a five-point scale, where 1 = very thin to 5 = obese (10)]. For this purpose, cows were dried off at 80 to 120 d before calving and were supplemented with concentrate ( 6 to 8 kg/d of DM per cow) plus corn silage (ad libitum intake) at pasture. One month before calving, cows were housed in individual stalls; cows were offered corn silage for ad libitum intake, formaldehyde-treated soybean meal ( 1 kg/d of DM per cow), soybean meal (0.5 kg/d of DM per cow), and various amounts of concentrate according to energy requirements of cows during this period (0.5 to 2.5 kg/d of DM per cow from 4 to 1 wk precalving). Feed was distributed at 0900 h. After calving, cows were fed a diet containing corn silage, formaldehyde-treated soybean meal, and soybean meal in the ratio of 75:20:5 on a DM basis, respectively, to maintain cows in negative energy balance throughout the experimental period. The proportion of soybean meal included in the diet was calculated such that protein requirements would be covered from approximately the 5th wk of lactation onward. Liver samples ( ∼600 mg) were obtained by biopsy once from all dry cows and from each of the 8 lactating cows on wk 1 ( d 4 ± 1), wk 2 ( d 11 ± 1), wk 4 ( d 25 ± 1), and wk 12 (between d 71 and 89) postcalving. Liver biopsy samples were rinsed in sterile saline solution, frozen in liquid N2, and stored at –80°C. Chemical Analysis Total lipids of liver samples (100 mg) were extracted in chloroform and methanol (2:1, vol/vol) and determined gravimetrically as previously described (26). Phospholipids were determined from total lipid extract by colorimetry after mineralization of organic P (26). Triglycerides were analyzed from total lipids according to the following method. After elimination of phospholipids absorbed on silicic acid, TG were saponified by 4N KOH and ethanol, followed by neutralization with 4N HCl, and then centrifugation. The free glycerol that was released in the supernatant was determined enzymatically using a triglyceride test kit (PAP 1000; BioMerieux, Charbonnie`res-lesBains, France). Total cholesterol from total lipid extract was determined enzymatically using a reagent kit (CHOD-iodide; Merck, Darmstadt, Germany). Extraction of hepatic proteins was performed on liver samples (50 mg) that were solubilized by addition of ice-cold lysis buffer [0.15 M NaCl, 5 mM Na2EDTA, 50 mM Tris (pH 7.4), 62.5 mM sucrose, 5 ml/ L of Triton X-100, and 5 ml/L of sodium deoxycholate]

HEPATIC GENE EXPRESSION OF APOLIPOPROTEIN B

containing 50 mg/ml of leupeptin, 50 mg/ml of pepstatin A, 1 mM of benzamidine, and 0.86 mM of phenyl sulfonyl fluoride (Sigma Chemical Co., St. Louis, MO). Hepatocytes were lysed using a Dounce homogenizer (PolyLabo, Strasbourg, France) and incubated overnight at 4°C on a rocking platform. After centrifugation for 20 min at 12,000 × g at 4°C, total proteins in the supernatant were determined by the method of Lowry (27). Concentrations of albumin and apo B in hepatocytes were evaluated from three aliquots of total protein extracts (20, 40, and 60 mg ) by Western blot analysis on polyacrylamide gel slabs at 10% (for albumin determination) or on continuous polyacrylamide gradient gel slabs from 3.5 to 7.5% (for apo B determination) in denaturing conditions according to Laemmli (25). The running buffer contained 0.025 M Tris·HCl (pH 8.3) and 0.192 M glycine. Proteins were then transferred onto polyvinylidene difluoride membranes (Immobilon PVDL; Millipore, Bedford, MA) by using Bio-Rad trans-cells (Bio-Rad, Hercules, CA) at 100 V for 90 min. Membranes were blocked by washing for 2 h in Trisbuffered saline (0.020 M Tris and 0.5 M NaCl) containing 50 g/L of milk powder and then incubated overnight with a rabbit antisera to bovine apo B purified from bovine LDL ( 2 ) or with a rabbit antisera to bovine albumin solution (1:10,000 and 1: 50,000, respectively) in Tris-buffered saline containing 5 ml/L of Tween ( TTBS) and 10 g/L of milk powder. Finally, membranes were washed three times in TTBS and incubated for 1 h with an anti-rabbit IgG linked with horseradish peroxidase, which was diluted (1:20,000) in TTBS and 10 ml/L of milk. After three washings in TTBS, antibodies were revealed by a light-based detection system using the ECL Western blotting kit (Amersham International, Bucks, United Kingdom). Membranes were exposed to multipurpose hyperfilm (Amersham International), and the signals were quantified by densitometric analysis of autoradiographies (Hoefer Gs 300; Hoefer Scientific Instruments, San Francisco, CA). Extraction of total RNA was performed from liver samples (200 mg) using the method of Chirgwin et al. (12). A 300-mg aliquot of each RNA preparation was incubated with DNase I (0.16 IU/mg of RNA) (Boehringer Mannheim, Meylan, France) for 45 min at 26°C and then for 20 min at 37°C with proteinase K (0.6 mg/ml; Boehringer Mannheim). After extraction of total RNA with phenol and chloroform (1:0.2, vol/vol), the integrity of all RNA samples was verified by electrophoresis on agarose gel slabs (10 g/L) and visualization of 18S and 28S ribosomal RNA stained with 0.025 M ethidium bromide. Total RNA abun-

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dance was measured with a spectrophotometer (Philips PU 8620; Elvetec, Cournon, France) using the conversion 1 absorbance unit = 40 mg/ml at 260 nm. The specificity of the cDNA probe corresponding to the nucleotide 9479 and to the nucleotide 10564 of human apo B gene (provided by G. D’Onofrio, Institut National de Recherche Me´dicale U 321, Paris, France) for bovine apo B mRNA and the integrity of isolated apo B mRNA were assessed by Northern blot as described by Maniatis et al. (28). Briefly, the 60 mg of total RNA that were extracted from each liver biopsy were denatured for 5 min at 65°C in a running buffer [10 mM Na2HPO4, 1 mM Na2-EDTA, and 5 mM sodium acetate (pH 7.0)] containing 200 ml/L of formamide and 60 ml/L of formaldehyde and then run on a denaturing (60 ml/L of formaldehyde) agarose gel (7.5 g/L) in the running buffer. Transfer of RNA onto nylon membranes (Genescreen; Biotechnology Systems, Boston, MA) was achieved for 3 h at 4°C by electroblotting using 20 mM Tris (pH 7.8), 10 mM sodium acetate, and 0.5 mM Na2-EDTA. Finally, membranes were irradiated for 30 s with UV light (365 nm) to fix RNA. Hybridization assays for determination of hepatic apo B mRNA were carried out by dot blot analysis of total RNA as described by Maniatis et al. (28). Aliquots of total RNA (60 mg ) extracted from each liver sample were denatured for 15 min at 68°C in 500 ml/ L of formamide, 175 ml/L of formaldehyde, and 1× SSC [0.015 M sodium citrate and 0.15 M NaCl (pH 7)]. Denaturated RNA then were blotted on nylon membranes (Genescreen; Biotechnology Systems) with successive decreasing dilutions (30 to 3 mg ) using the dot blot apparatus (Schleicher and Shull, Ecquevilly, France). The RNA were fixed by UV light. The cDNA probe of human apo B (1.2 kb) was labeled with deoxycytidine 5′-[a32]triphosphate ( [a32P]dCTP) (Amersham International) using a random priming labeling kit (Boehringer Mannheim). The oligonucleotide of rat 18S RNA (Eurogentec, Angers, France) was labeled with [g-32P]ATP according to the following method as described by Maniatis et al. (28). Briefly, 50 ng of the oligonucleotide were incubated 45 min at 37°C with 50 mCi of [g-32P]ATP and 8 IU of T4 polynucleotide kinase (Boeringer Mannheim) in solution of 70 mM Tris·HCl (pH 7.6), 10 mM MgCl2, and 5 mM dithiothreitol. Blots of RNA were prehybridized for 2 h and then hybridized for 18 h at 42°C in 200 ml/L of formamide with a 3× solution of SSP-EDTA [0.45 M NaCl, 0.03 M NaH2PO4, and 3 mM Na2-EDTA (pH 7.4)], 10 g/L of SDS, 5× Denhart Journal of Dairy Science Vol. 80, No. 4, 1997

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GRUFFAT ET AL. TABLE 1. Mean BW, body condition score, and milk production of lactating cows during the first 12 wk of lactation. Lactation

BW, kg X SE Body condition score1 X SE Milk production, kg/d X SE

wk 1 (n = 8)

wk 2 (n = 8)

wk 4 (n = 8)

wk 12 (n = 8)

768.5a 16.1

744.7b 45.0

695.2c 10.3

677.5c 17.8

3.62a 0.16

3.28b 0.18

2.72c 0.24

35.7ad 1.9

40.5bc 2.2

38.1ac 1.3

2.03d 0.32 32.4d 1.6

a,b,c,dComparison 1Five-point

between different weeks of lactation by Student’s t test ( P < 0.01). scale, where 0 = very thin to 5 = obese (10).

(20 g/L of BSA, 20 g/L of ficoll, and 20 g/L of polyvinylpyrolidone) in the presence of salmon sperm DNA (0.1 mg/ml). Membranes were washed four times at 20°C for 5 min in 2× SSC and 1 g/L of SDS, followed by two more washings at 55°C for 20 min in 1× SSC with 1 g/L of SDS. Hybridization of RNA with a cDNA probe was revealed by autoradiography at –80°C for 24 h using multipurpose hyperfilms (Amersham International), and the signals were quantified by densitometric analysis (densitometer Ultroscan with GSW 365 software; Hoefer Scientific Instruments, San Francisco, CA). The determination of hepatic DNA content was performed according to the method described by

Labarca and Paigen (24). Samples of hepatic tissue (50 mg) were solubilized by addition of 5 ml of icecold lysis buffer [0.05 M NaPO4, (pH 7.4), 2 M NaCl, and 2 mM Na2-EDTA]. The DNA determinations were performed in PBS (0.05 M NaPO4 and 2 M NaCl; pH 7.4) containing 0.2 mg/ml of bisbenzimidazole (Hoechst 33258; Sigma-Aldrich, Milwaukee, WI). The DNA content was determined by fluorometric analysis, and excitation and emission wavelengths were 356 nm and 458 nm, respectively, using calf thymus DNA ( 5 mg/ml) as a DNA reference (SigmaAldrich). The number of hepatocytes present in each liver sample was calculated from values of DNA; 106 hepatocytes contained 6.4 mg of DNA (38).

TABLE 2. Mean concentrations of the major lipids in the liver of dry (pregnant or nonpregnant) and lactating cows. Dry cows Component

Pregnant (n = 5)

Nonpregnant (n = 4)

Lactating cows ( n = 8 ) wk 1

wk 2

wk 4

wk 12

(mg/g of fresh liver) Total lipids X SE Triglycerides X SE Phospholipids X SE Total cholesterol X SE A,BComparison a,b,cComparison

24.86A 3.09

29.25A 2.71

79.50a,B 7.00

127.90b,B 15.50

97.50ab,B 20.70

32.60c,A 1.70

3.71A 1.08

3.42A 6.60

44.20a,B 6.60

95.70b,B 15.90

66.40ab,B 20.80

3.90c,A 0.70

22.20A 1.88

19.82A 2.64

31.30a,B 2.50

28.30b,B 0.80

27.50b,A 1.30

25.70b,A 1.80

1.64 0.19

2.42 0.44

2.50a 0.20

2.50a 0.20

2.20a 0.20

1.80b 0.10

between dry and lactating cows by Mann-Whitney’s test (P < 0.01). between different weeks of lactation by Student’s paired t test ( P < 0.01).

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HEPATIC GENE EXPRESSION OF APOLIPOPROTEIN B TABLE 3. Mean hepatic concentrations of total RNA, total proteins, and albumin in dry (pregnant or nonpregnant) and lactating cows. Dry cows Pregnant (n = 5)

Component

Lactating cows ( n = 8 )

Nonpregnant (n = 4)

wk 1 (mg/10 6

Total RNA X SE Total proteins X SE

2.53 0.20 151A 5

Albumin X SE

2.52 0.09

37.50 9.20

wk 4

wk 12

cells)

2.88 0.09

146A 2

34.80 3.10

wk 2

2.80 0.08

2.68 0.11

2.80 0.07

176ab,B 173b,A 5 6 (AU 1/106 cells)

173b,A 6

191a,B 3

36.10 2.80

39.70 6.10

32.10 5.90

32.20 5.90

A,BComparison

between dry and lactating cows by Mann-Whitney’s test (P < 0.01). between different stages of lactation by Student’s paired t test ( P < 0.01). 1Arbitrary units. a,bComparison

Statistical Analysis Comparisons of results between independent groups (dry cows vs. pregnant cows vs. lactating cows in wk 1, 2, 4, or 12) were carried out using the nonparametric Mann-Whitney test (29). The effect of week of lactation was studied using Student’s paired t test. RESULTS Characteristics of BW, body condition score, and milk production of lactating cows during wk 1, 2, 4, and 12 are given in Table 1. The cows suffered major losses of BW and body condition between wk 1 and 12 and especially during wk 1 to 4, when 9.5% of their BW ( P < 0.01) was lost. Mean body condition score

declined by 0.9 units (approximately 25.0% change; P < 0.01) between wk 1 and 4. Milk production, however, remained relatively constant during the first 4 wk of lactation but decreased by 15.0% ( P < 0.01) between wk 4 and 12 of lactation. Compared with the dry (pregnant or nonpregnant) cows, total lipid content of the liver of underfed, fat lactating cows was approximately 3-fold higher ( P < 0.01) at wk 1 of lactation (Table 2). Hepatic lipid content was maximal at wk 2, then declined 24% by wk 4, and was at 74.5% ( P < 0.01) by wk 12, when values were similar to those observed for dry cows. Lipid accumulation in the liver, noted during the first 4 wk of lactation, resulted mainly from a large increase in TG (approximately a 28.0-fold increase in wk 2 compared with that of dry cows; P < 0.01) and a

TABLE 4. Mean hepatic concentrations of apolipoprotein (apo) B and its mRNA in dry (pregnant or nonpregnant) and lactating cows. Dry cows Pregnant (n = 5)

Nonpregnant (n = 4)

Lactating cows ( n = 8 ) wk 1

wk 2

wk 4

wk 12

(AU 1/106 cells) apo B mRNA X 35.79 SE 7.88 apo B X 81.26A SE 14.80

33.45 4.80

23.95 3.15

25.97 4.35

27.54 3.73

30.23 3.02

76.16A 21.01

25.06a,B 3.37

30.90a,A 15.07

34.98a,A 4.90

93.66b,A 24.14

A,BComparison

between dry and lactating cows by Mann-Whitney’s test (P < 0.01). between different wks of lactation by Student’s paired t test ( P < 0.01). 1Arbitrary units. a,bComparison

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smaller increase in phospholipids (approximately a 1.6-fold increase in wk 1 compared with dry cows; P < 0.01). The TG fraction only represented 11.7 and 14.9% of total lipids in hepatocytes of dry cows (pregnant or nonpregnant, respectively) but became the main lipid component in the liver of underfed, fat cows during the first 4 wk of lactation (55.6 to 74.8% of total lipids). Conversely, no significant differences in cholesterol content of hepatocytes occurred among different groups of cows (Table 2). Potential indicators of hepatic synthesis of protein, such as hepatic concentrations of total RNA, total proteins, and albumin, are presented in Table 3. The physiological state of the cows had no effect on either total RNA or albumin levels in hepatocytes. However, the total content of hepatic protein increased during the first 12 wk of lactation for the underfed, fat cows,

which was higher at wk 1 and 12 ( P < 0.01) than that of dry cows. From results of the Northern blot analysis, hybridization of the cDNA probe of human apo B to total hepatic RNA showed a single band corresponding to apo B100 mRNA (14 kb) in all cows (Figure 1A). This result indicated the high specificity of this cDNA probe for bovine apo B mRNA and the complete integrity of analyzed apo B mRNA. Similar amounts of total RNA were loaded on agarose gel for electrophoresis; the bands of 18S RNA that were detected on autoradiography were comparable (Figure 1B). The Northern blot analysis showed no significant variation between apo B mRNA levels of dry cows and lactating cows (Figure 1A) for representative cows of the different physiological periods. However, dot blot analysis determined mRNA more precisely because this method integrated values of four successive dilutions of the same RNA sample (Figure 1C) from the same representative cows used for Northern blot analysis. The dot blot method showed that hepatic apo B mRNA was lower for 7 of 8 underfed, fat cows at wk 1 of lactation compared with that of dry cows (Table 4). For the underfed, fat cows during the first 12 wk of lactation, hepatic apo B mRNA tended to increase ( P < 0.1) regularly between wk 1 and 12 (Table 4). Intrahepatic concentrations of apo B were determined by Western blot analysis using a serum against bovine apo B as illustrated in Figure 2 from representative cows at different physiological stages. This method detected a single complementary band of apo B with a molecular mass of approximately 520 kDa, corresponding to apo B100 in all bovine hepatic samples (Figure 2). Hepatic concentrations of apo B were considerably lower for underfed, fat cows at wk 1 of lactation (3-fold lower; P < 0.01) than for dry cows (Table 4 ) but increased between wk 4 and 12 of lactation by 63% ( P < 0.01) to achieve values similar to those recorded in dry cows. By regression analysis, a negative correlation was observed ( r = –0.638; P < 0.01) between hepatic apo B and TG concentrations for underfed, fat cows between wk 1 and 12 of lactation (Figure 3). This correlation was due to changes in stage of lactation, but not to variations among cows for a given stage of lactation. DISCUSSION

Figure 1. Determination of levels of hepatic apolipoprotein (apo) B mRNA of dry (pregnant or nonpregnant) and lactating cows (one representative cow per physiological state) by Northern blot ( A and B ) and dot blot ( C ) analysis. Hybridizations were performed with the 32P-labeled human apo B cDNA probe ( A and C ) and with the 32P-labeled rat 18S RNA oligonucleotide ( B ) . Journal of Dairy Science Vol. 80, No. 4, 1997

Expression of Apo B Gene in the Liver of Cows In this experiment, variations of gene expression of apo B in the liver of cows during the cycle from gestation to lactation were analyzed. To our

HEPATIC GENE EXPRESSION OF APOLIPOPROTEIN B

knowledge, this experiment was the first time that hepatic levels of apo B and the corresponding mRNA and TG had been simultaneously measured in ruminants. However, rates of synthesis and degradation of apo B mRNA and apo B were not determined. The reliability of determination of apo B mRNA in the bovine liver by Northern blot analysis has also been reported. Indeed, the sequence of the human apo B cDNA that was used as a probe for determination of apo B mRNA in cows corresponds to the domain of apo B that binds the LDL receptor, which is highly conserved among species. This analytical approach— using hybridization of the cDNA probe to hepatic bovine RNA transferred onto nylon membranes— revealed, in all hepatic samples, only a single complementary mRNA species with a mean length of 14 kb, which was similar to that found for other species ( 8 ) . Intrahepatic apo B was determined according to the procedure of Western blot using an antisera to calf apo B raised in rabbits in our laboratory. Regardless of the physiological state of cows, whether dry (pregnant or nonpregnant) or in early lactation, only one single band was identified on nylon filter; the band corresponded to apo B with a molecular mass of 520 kDa. Based on a comparison of data obtained for bovine apoproteins in plasma and intestinal lymph ( 3 ) , this band corresponds to apo B100. Indeed, two major molecular mass forms of apo B are synthesized in the body, a larger form, apo B100 (520 kDa), and a smaller form, apo B48 (265 kDa) ( 4 ) . In mammals, a single gene codes for both forms, but apo B48 is produced by editing via a posttranscriptional modifi-

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Figure 3. Correlation between hepatic apolipoprotein (apo) B and triglyceride ( T G ) concentrations determined from fat, underfed cows at wk 1, 2, 4, and 12 of lactation. ( n = 32; r = –0.638; P < 0.01). AU = Arbitrary units.

cation of the apo B mRNA (13). In the bovine, using primer extension of an analyses of reverse-transcribed polymerase chain reaction, the editing process of apo B mRNA has been demonstrated to be almost complete (<94%) in the small intestine, but totally absent in the liver (18). The existence of only apo B100 in hepatocytes of cows confirms the absence of the editing process shown in the bovine liver. Induction of Fatty Liver in High Producing Dairy Cows

Figure 2. Determination of levels of hepatic apolipoprotein (apo) B of dry (pregnant or nonpregnant) and lactating cows (one representative cow per physiological state) by Western blot analysis by using a specific rabbit antiserum to bovine apolipoprotein (apo) B.

Fatty liver is considered to be a peripartum metabolic disorder that arises from excessive FA mobilization when energy intake is insufficient to support maintenance and milk production. Numerous researchers (21, 31, 34) reported that, for cows fed to achieve high body condition at calving, liver lipids were two to three times higher within 1 to 2 wk of calving than those of cows fed to achieve thin to normal body condition. In the present experiment, high producing dairy cows were overfed during gestation and then underfed after parturition to increase fat mobilization and to induce hepatic steatosis. Under these conditions, the variations of lipid concentrations in the hepatocytes were typical of those for cows in early lactation as reported by Grummer (21); fatty liver was characterized by mean hepatic TG concentrations of around 50 to 100 mg/g during wk 2 of lactation, as previously reported (31). However, 1 mo before calving, cows hepatic TG concentrations were Journal of Dairy Science Vol. 80, No. 4, 1997

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similar to those of nonpregnant dry cows, indicating that, at 1 mo before calving, TG infiltration of the liver had not yet commenced. The TG content of the liver of cows was low during early gestation but doubled between 17 d prepartum and calving (5, 37), suggesting that hepatic TG accumulation commenced from wk 4 to 3 before parturition. Lipid infiltration of the liver has been reported (15, 22, 30) to be negatively correlated with circulating concentrations of apo B in cows during early lactation. Those researchers associated the low plasma apo B concentrations with the low liver concentrations of apo B mRNA in cows that developed fatty liver 6 d postpartum, as reported earlier by Cardot et al. ( 7 ) . A failure in apo B synthesis might be directly linked to the development of fatty liver (31). Moreover, for cows with a severe infiltration of lipids into the liver, alterations in the ultrastructural characteristics of hepatocytes, including increased cell volume, mitochondrial damage, and decreased volume of rough endoplasmic reticulum, have also been reported (35). Furthermore, plasma concentration of albumin decreased for cows with fatty liver, suggesting a major alteration in the synthesis of liver proteins (36). In the present experiment, no significant variations were recorded for total RNA, total proteins, or albumin concentrations in the liver of cows, which suggests that hepatocytes were probably not significantly altered by a relatively moderate infiltration of lipids during the 1st mo of lactation. However, hepatic apo B mRNA decreased slightly for cows during the initial weeks of lactation compared with that of dry cows (pregnant or nonpregnant) or cows in midlactation (12 wk). This result was in agreement with previous results for cows ( 7 ) and with the few reports on HepG2 cells or on rat liver, suggesting an acute control of mRNA synthesis or degradation during different metabolic states ( 1 ) . Hepatic apo B was much lower during the initial weeks of lactation than during dry (pregnant or nonpregnant) and midlactation periods. These results indicate a specific regulation of apo B synthesis during early lactation in cows. The fact that the decrease of apo B concentration was much higher than the decrease of apo B mRNA concentrations tends to suggest either a posttranscriptional regulation or an increased secretion of VLDL. However, Durand et al. ( 1 5 ) have demonstrated that total plasma concentrations of apo B and apo Blipoproteins were also lower in these fat, underfed high producing dairy cows during wk 1 of lactation than those during wk 12. Moreover, during the first 2 wk of lactation, hepatic secretion of VLDL is low in Journal of Dairy Science Vol. 80, No. 4, 1997

cows because hepatic balance of VLDL is negative (14). In HepG2 line cells, the rate of apo B mRNA translation can be decreased by insulin ( 1 ) . In the present experiment, the decrease of intrahepatic apo B in high producing dairy cows in early lactation might not be explained by an insulin-modulated translation of apo B because insulin was low in these cows (around 6 mU/ml of plasma) (11). In rats with protein deficiencies, the formation of lipoproteins also can be altered to favor lipid infiltration of the liver (16). Under such conditions of low dietary protein, the low secretion of VLDL is associated with low plasma VLDL apo B, which may be attributable to the reduction in the synthesis of apolipoproteins ( 6 ) . Conversely, Durand et al. ( 1 4 ) have previously reported that infusion of L-methionine plus L-lysine into the portal veins of lactating dairy cows favored the apparent production of VLDL by the liver. This result suggested a potential role of these limiting amino acids in the regulation of hepatic VLDL secretion. Protein degradation in the liver might be increased during amino acid restriction (39). Many in vitro studies with rat hepatocytes and HepG2 cells have suggested that a significant proportion of newly synthesized apo B is rapidly degraded and fails to assemble into lipoprotein particles. Intracellular apo B degradation is stimulated by insulin and decreased by high concentrations of intracellular TG, cholesteryl esters, and phospholipids ( 1 ) . Protection of apo B from intrahepatic catabolism should occur during steatosis in cows during early lactation because, during this period, insulinemia was low ( 1 1 ) and lipids accumulated in hepatocytes. Conversely, the present results tended to suggest that the increased degradation of apo B probably resulted from either an ultrastructural change in the characteristics of hepatocytes resulting from the infiltration of hepatocyte by lipids ( 3 5 ) or a limited availability of specific amino acids during this period or this physiological state of early lactation. CONCLUSIONS To our knowledge, the present experiment is the first time that simultaneous measurements have been made of hepatic apo B, its corresponding mRNA, and liver TG of cows during the cycles of gestation and lactation. Hepatic steatosis in cows during early lactation appeared to be associated with a dramatic decrease in intrahepatic apo B and a slight decline in apo B mRNA, indicating preferential posttranscriptional regulation of apo B synthesis. This regulation

HEPATIC GENE EXPRESSION OF APOLIPOPROTEIN B

could be attributable either to a reduction in the translation of proteins as a consequence of feed intake limitation of cows in early lactation or to an increase in intracellular degradation of apo B or to an increase in hepatic VLDL secretion. More experiments are required to test these different hypotheses and to identify the limiting factors that control intrahepatic recruitment of lipids and its coordination with apo B synthesis. ACKNOWLEDGMENTS We thank G. D’Onofrio for providing a cDNA probe of human apo B, J. Lefaivre for surgical preparation of the cows, A. Ollier for cow management, and F. Duboisset, C. Legay, and M. Martinaud for technical assistance. REFERENCES 1 Adeli, K., A. Mohammadi, and J. Macri. 1995. Regulation of apolipoprotein B biogenesis in human hepatocytes: posttranscriptional control mechanisms that determine the hepatic production of apolipoprotein B-containing lipoproteins. Clin. Biochem. 28:123. 2 Auboiron, S., D. Durand, P. M. Laplaud, D. Levieux, D. Bauchart, and M. J. Chapman. 1990. Determination of the respective density distributions of low and high-density lipoprotein particles in bovine plasma and lymph by immunoassay of apoproteins A-I and B. Reprod. Nutr. Dev. Suppl. 2: 227S. 3 Bauchart, D. 1993. Lipid absorption and transport in ruminants. J. Dairy Sci. 76:3864. 4 Bauchart, D., D. Gruffat, and D. Durand. 1996. Lipid absorption and hepatic metabolism in ruminants. Proc. Nutr. Soc. 55: 39. 5 Bertics, S. J., R. R. Grummer, C. Cadorniga-Valino, and E. E. Stoddard. 1992. Effect of prepartum dry matter intake on liver triglyceride concentration and early lactation. J. Dairy Sci. 75: 1914. 6 Bouziane, M., J. Prost, and J. Belleville. 1993. Unsaturated fatty acid bioavailability in growing rats fed low or adequate protein diets with sunflower or soybean oils. J. Nutr. Biochem. 4:399. 7 Cardot, P., A. Mazur, M. Pessa, J. Chambaz, and Y. Rayssiguier. 1988. Expression he´patique du ge`ne d’apolipoprote´ine B chez la vache au cours de la lactation. Reprod. Nutr. Dev. 28: 169. 8 Chan, L. 1992. Apolipoprotein B, the major protein component of triglyceride-rich and low density lipoproteins. J. Biol. Chem. 267:25621. 9 Chilliard, Y. 1993. Les adaptations me´taboliques et le partage des nutriments chez l’animal en lactation. Page 431 in Biologie de la Lactation. J. Martinet and L. M. Houdebine, ed. Inst. Natl. Rech. Med. and Inst. Natl. Rech. Agron. Publ., Paris, France. 10 Chilliard, Y., M. Cisse´, R. Lefaivre, and B. Re´mond. 1991. Body composition of dairy cows according to lactation stage, somatotropin treatment, and concentrate supplementation. J. Dairy Sci. 74:3103. 11 Chilliard, Y., A. Ollier, D. Durand, R. Lefaivre, M. Tourret, D. Thomas, E. Girard, G. Sauvage, D. Gruffat, J. C. Robert, P. Williams, and D. Bauchart. 1994. Body lipid mobilization, acetonemia and hepatic steatosis in the underfed high yielding dairy cow during early lactation. Ann. Zootech. (Paris) 43(Suppl. 1):46S.

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