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Article

Mammary Lipogenic Enzyme Activity, trans Fatty Acids and Conjugated Linoleic Acids Are Altered in Lactating Dairy Cows Fed a Milk Fat–Depressing Diet¹

Liliana S. Piperova^*, Beverly B. Teter, Israel Bruckental^**, Joseph Sampugna, Scott E. Mills, Martin P. Yurawecz, Jan Fritsche, Ken Ku and Richard A. Erdman^*2

^* Department of Animal and Avian Sciences and Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742; ^** Volcani Institute, Bet Dagan, Israel 50-250; Department of Animal Sciences, Purdue University, West Lafayette, IN 47907 and U.S. Food and Drug Administration, Center for Food Safety and Applied Nutrition, Washington, D.C. 20204

²To whom correspondence should be addressed.

	ABSTRACT

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The objectives of the present study were to examine the effect of a milk fat–depressing (MFD) diet on: 1) the activity of mammary acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS), 2) ACC mRNA relative abundance and 3) distributions of conjugated linoleic acids (CLA) and trans-18:1 fatty acids (tFA) in milk fat. Twelve lactating Holstein cows were used in a single reversal design. Two diets were fed: a control diet (60:40% forage/concentrate) and an MFD diet (25:70% forage/concentrate, supplemented with 5% soybean oil). The MFD diet decreased (P < 0 0.001) milk fat by 43% and ACC and FAS activity by 61 and 44%, respectively. A reduced ACC mRNA relative abundance (P < 0.001) corresponded with the lower ACC activity. The fatty acids synthesized de novo were decreased (P < 0.002), whereas tFA were increased from 1.9 to 15.6% due predominantly to a change in trans-10-18:1 isomer (P < 0.001). With the MFD diet, the trans-7, cis-9 and trans-10, cis-12 CLA isomers were elevated (P < 0.001), in contrast to the decrease in trans-11-18:1 (P < 0.001) and cis-9, trans-11-18:2. The data were consistent with a dietary effect on mammary de novo FA synthesis mediated through a reduction in ACC and FAS activity and in ACC mRNA abundance. The results were compatible with a role of trans-10, cis-12 CLA in milk fat depression, but alterations noted in tFA and other CLA isomers suggest that they also may be important during diet-induced milk fat depression.

KEY WORDS: • acetyl-CoA carboxylase • fatty acid synthase • trans fatty acids • conjugated linoleic acid • lactating dairy cows

	INTRODUCTION

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In lactating dairy cows, various dietary conditions (Grummer 1991 , Kalscheur et al. 1997a , Selner and Shultz 1980 ) result in milk fat depression. A reduction in milk fat content has also been associated (Chouinard et al. 1999a , Loor and Herbein 1998 , Romo et al. 1996 ) with conjugated linoleic acids (CLA)³ and trans-18:1 fatty acids (tFA), produced during rumen biohydrogenation of polyunsaturated fatty acids (PUFA) (Harfoot et al. 1973 , Tanaka and Shigeno 1976 ).

Using feeding (Kalscheur et al. 1997a and 1997b ) and infusion (Gaynor et al. 1994 and 1995 , Romo et al. 1996 ) experiments with lactating cows, we were able to demonstrate that an increase in milk tFA was associated with milk fat depression. However, the lack of a consistent reduction in milk fat despite an elevation in total tFA (Griinari et al. 1997 , Kalscheur et al. 1997b ) led Griinari et al. (1997) to suggest a role of specific trans-isomers in milk fat depression. Subsequently, Griinari et al. (1998) reported that an increase in the trans-10-18:1 isomer was associated with milk fat depression. Although we did not emphasize a connection, we observed a decrease in milk fat and an increase in the trans-10 and trans-12-18:1 isomers in milk of lactating cows fed a milk fat–depressing (MFD) diet (Piperova et al. 1997 ).

The abomasal infusion of commercially available CLA mixtures has also resulted in reduced milk fat in lactating cows (Chouinard et al. 1999a , Loor and Herbein 1998 ). Recently, Baumgard et al. (2000) provided convincing data that the trans-10, cis-12 CLA isomer is responsible for milk fat depression. Nevertheless, a decrease in milk fat was also observed when a CLA mixture containing the cis-8, trans-10 isomer (Chouinard et al. 1999b ) was infused, suggesting that other CLA isomers may also be involved. Detailed studies of CLA isomer distribution may shed further light on this subject.

During dietary milk fat depression, fatty acids (FA) synthesized de novo in the mammary gland were disproportionately reduced (Banks et al. 1984 , Loor and Herbein 1998 , Wonsil et al. 1994 ), suggesting possible effects on lipogenic enzyme activity. Acetyl-CoA (ACC) carboxylase is a likely candidate because it is the rate-limiting enzyme for de novo FA synthesis (Mellenberger et al. 1973 ) and has been reported to be inhibited by long chain FA (Wakil et al. 1983 ). Also, dietary PUFA affect the gene expression of various lipogenic enzymes (Clarke 1993 , Clarke and Jump 1994 ) and have been shown (Toussant et al. 1981 ) to coordinately suppress the hepatic content of FAS and ACC proteins. If specific tFA and/or CLA isomers are involved in reducing de novo milk FA synthesis, it is possible that they exert effects similar to those described above.

In view of this, we wondered whether an MFD diet would result in increases in specific CLA and tFA isomers in milk fat and in decreases in mammary ACC and FAS activity and ACC mRNA relative abundance. Consequently, the objectives of the present study were to determine the effect of an MFD diet on the activities of ACC and FAS and relative abundance of ACC mRNA in cow mammary gland and to examine the milk CLA and trans-18:1 isomer profiles during milk fat depression.

	MATERIALS AND METHODS

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Animals and diets.

All procedures for this study were carried out under Protocol R-95-33A approved by the University of Maryland Animal Care and Use Committee. Twelve lactating Holstein cows (84 ± 13 d postpartum) were used. The control diet had a forage/concentrate ratio of 60:40 and was formulated to meet the NRC requirements for milk production at 40 kg/d and 3.5% milk fat. Cows were fed the control diet during the 2-wk preliminary period and randomly assigned to either the control or an MFD diet that contained 25% forage and 70% concentrate, supplemented with 5% soybean oil (Wesson vegetable oil, ingredient: soybean oil). Diets were fed in a single reversal design during 3-wk experimental periods.

The ingredients and chemical composition of the diets are presented in Table 1 . Forage and concentrate dry matter were determined weekly, and the total mixed ration was adjusted accordingly to maintain a constant forage/concentrate ratio on a dry matter basis during the experiment. Cows were housed in tie stalls and bedded with wood shavings except when they were turned out to be milked in a milking parlor at 0200 and 1400 h. Diets were fed as total mixed ration once daily at 0800 h. Feed refusals were recorded once daily at 0600 h.

Milk production was recorded daily, and milk samples were collected for six consecutive milking sessions at the end of each experimental period. Milk fat, protein and somatic cell count were analyzed with infrared analysis (Foss Milkoscan; Foss Food Technology Corp., Eden Prairie, MN). FA composition of milk fat was determined as described below.

Total FA pattern.

FA methyl esters (FAME) were prepared according to a direct transesterification method with anhydrous methanolic HCl (Gaynor et al. 1995 ). Resulting FAME were separated and quantified with a 30 m x 0.25 mm fused silica capillary column coated with SP-2380 (Supelco Inc., Bellefonte, PA), essentially as described by Wong et al. (1993) . A variety of FAME standard mixtures, including GLC-60 (Nu Check Prep; Elysian, MN), were used to help identify components and assist in the calculation of response factors. Correction factors for total trans-18:1 values were obtained as described previously (Atal et al. 1994 , Sampugna et al. 1982 ).

Short and medium chain FA were analyzed as butyl esters (FABE), which were mathematically converted to FAME and normalized to the FAME chromatogram (Gander et al. 1962 ). The original FABE procedure was modified as follows. Milk samples (200 µL), in screw-capped test tubes, were heated at 100°C for 1 h in the presence of 1 mL of butanol and 200 µL of acetyl chloride. Aliquots of the upper layer were analyzed using a Hewlett-Packard 5880 gas-liquid chromatograph equipped with a split injector, a flame ionization detector and a 25 m x 0.2 mm fused silica capillary column coated with HP1 (Hewlett Packard, Avondale, PA). Helium was used as the carrier gas at a flow rate of 2 mL/min with a split ratio of 45:1. After 5 min at 90°C, the column temperature was raised (4°C/min) to 106°C, and at 10 min, it was programmed at 5°C/min to a final temperature of 250°C. Standard mixtures, including GLC-60, were converted to FABE to aid in the identification and quantification of components.

trans-18:1 isomer distribution.

Butyl esters, prepared as described above, were separated by preparative Ag⁺-thin layer chromatography to obtain a trans monoene fraction (Sampugna et al. 1982 ) using FABE standards of 14:0, cis-9-18:1 and trans-9-18:1 to help locate fractions of interest. A GLC system similar to that previously described (Wong et al. 1993 ) was used to separate isomers, except that a 100 m x 0.25 mm fused silica capillary column (SP-2560; Supelco Inc.) was used at a column temperature of 173°C and a split ratio of 100:1 and with nitrogen as make-up gas (25 mL/min). Isomers of cis and trans FA with double bonds in the 6, 7, 9, 11, 12, 13 and 15 positions (Sigma Chemical Co., St. Louis, MO), as well as a sample of trans-10-18:1 and cis- and trans-18:1 fractions isolated from a shortening sample (Sampugna et al. 1982 ), were converted to FABE to assist in the identification and quantification of the trans-18:1 isomers.

Conjugated FA.

The milk fat was extracted using a modified Folch procedure (Christie 1982 ). FAME were prepared as described by Sehat et al. (1998a) and were analyzed using GLC and Ag⁺–high performance liquid chromatography (HPLC) to obtain estimates of total CLA and to determine isomer distribution patterns. The GLC system, as described previously (Eulitz et al. 1999 ), used a fused silica capillary column (CP-Sil 88; 100 m x 0.25 mm i.d. x 0.2 µm film thickness; Chrompack), which was held at 70°C for 4 min after injection, temperature programmed (13°C/min) to 175°C, held at 175°C for 27 min and then temperature programmed (4°C/min) to 215°C and maintained at 215°C for 31 min. Hydrogen was used as the carrier gas, at a split ratio of 20:1. The detector and injector were set at 250°C.

Argentation–HPLC separation (Sehat et al. 1998b ) of CLA methyl esters was carried out with an HPLC (Waters 510 solvent delivery system; Waters Associates, Milford, MA), equipped with a 100-µL injection loop (Waters 600E System Controller), a photodiode array detector (Waters 996) operated at 233 nm and a Waters software program (Millennium Version 2.15). Three ChromSpher 5 Lipids analytical silver-impregnated columns (each 4.6 mm i.d. x 250 mm stainless steel, 5-µm particle size; Chrompack, Bridgewater, NJ) were used in series. The mobile phase was 0.1% acetonitrile in hexane and operated isocratically at a flow rate of 1.0 mL/min. The column head pressure was 1050 psi for the three columns in series. The flow commenced for 0.5 h before sample injection. Typical injection volumes were 5–15 µL, representing 40–120 µg of FAME. Details on the identification and quantification of the CLA isomers have been described elsewhere (Eulitz et al. 1999 , Sehat et al. 1998a and 1998b ).

Biopsy procedure.

Mammary tissue biopsy was performed (Farr et al. 1996 ) on 10 cows during the 3rd wk of each experimental period to obtain a core sample of 0.7–0.8 g. For the first three or four milking sessions after biopsy, care was taken to milk the cows thoroughly by hand to remove any blood clots lodged in the gland.

Enzyme analysis.

After the biopsy procedure, tissue samples were homogenized as described by Mellenberger et al. (1973) . The enzyme activities were assayed in the cytosolic fraction (105,000 x g) at 37°C in the linear range of activity. Acetyl-CoA carboxylase (EC 6.4.1.2) activity was determined using the ¹⁴C-labeled bicarbonate fixation method (Mellenberger et al. 1973 ) and expressed as nanomoles of bicarbonate incorporated into acid-stable products per minute per milligram of cytosolic protein. FA synthase activity was determined spectrophotometrically (Hardie et al.1981 ) by measuring malonyl-CoA–dependent oxidation of NADPH at 37°C and was expressed as nanomoles of NADPH oxidized per minute per milligram of cytosolic protein. Protein concentrations were measured according to the bicinchoninic acid procedure (Pierce Chemical, Rockford, IL) with bovine serum albumin as the standard.

Northern blot analysis of total RNA.

Immediately after biopsy, the mammary tissue (250 mg) was homogenized in 2.5 volumes of ice-cold denaturing solution (4 mol/L guanidinium thiocyanate, 25 nmol/L sodium citrate, pH 7, 0.5% sarcosyl, 0.1 mol/L 2-mercaptoethanol) and kept at -80°C until analyzed. The total RNA was isolated as described by Chomczynski and Sacchi (1987) and assessed for purity. Concentrations were determined spectrophotometrically at A₂₆₀. Only samples with A₂₆₀/₂₈₀ ratios of >1.80 were analyzed. The RNA integrity was confirmed visually on a 1% agarose-formaldehyde minigel. Total RNA (50 µg), separated as described by Sambrook et al. (1989), was transferred overnight to a positively charged nylon membrane (Hybond-N⁺; Amersham International, Buckinghamshire, U.K.) via capillary action. The RNA was cross-linked to the membrane (40 s at 1200 µJ x 100) in a UV Stratalinker 1800 (Stratagene, La Jolla, CA). The acetyl-CoA carboxylase cDNA (730 bp) was amplified from pig adipose tissue RNA, and the sequence corresponding to nucleotides 642-1370 of the rat ACC cDNA (Lopez-Casillas et al. 1988 ) was confirmed by Liu (1992) . The membrane was prehybridized (5–6 h) and hybridized (16–18 h with 2.45–2.95 x 10⁹ dpm/L [-³²P]dCTP-labeled ACC cDNA) in a solution consisting of 50% formamide, 50 mmol/L NaH₂PO₄, pH 6.5, 5 mmol/L EDTA, pH 7.4, 25% 20x standard saline citrate, pH 7.0, 5x Denhardt’s solution, 10% SDS and 200 µg of denatured salmon sperm DNA, except that the hybridization solution contained 1x Denhardt’s solution. Subsequently, the membrane was stripped in 5 g/L SDS at 100°C for 1 h and rehybridized with 25 ng of bovine ribosomal protein S4 (accession no. U31305; 638 bp) cDNA (kindly provided by Dr. S. L. Ogg, University of Maryland, College Park, MD) to correct for sample loading and transfer variation during Northern blotting.

Autoradiograms were obtained through the exposure of membranes to Kodak XAR-5 film for 7 d at -80°C. A single band of 9.5 kb for ACC mRNA was visible (Fig. 1 ), and the size was verified by an RNA marker ladder (0.24–9.5 kb; Sigma Chemical). Autoradiograms were scanned twice and analyzed with ImageQuant software (Molecular Dynamics, Sunnyvale, CA). The bovine ribosomal protein S4 mRNA was detected through exposure to Kodak XAR-5 film for 6 h at -80°C. Values were normalized across samples using bovine ribosomal protein S4 mRNA. The results are presented as the relative abundance of ACC mRNA in densitometric units.

View larger version (85K): [in this window] [in a new window]   Figure 1. A representative Northern blot of mammary tissue mRNA for acetyl-CoA carboxylase (ACC) from lactating cows fed a control or a milk fat–depressing (MFD) diet. Total RNA (50 µg) was hybridized to a [32P]-labeled ACC cDNA probe. The intensity of a single transcript (7-d exposure) of 9.5 kb was compared for both diets. The bovine ribosomal protein S4 cDNA (BRP) transcript was visualized after 6-h exposure.

Statistical analyses were conducted using the General Linear Models Procedure in the Statistical Analysis System (Version 6.12, 1998; SAS Institute, Cary, NC ). The statistical model included the effect of cow, experimental period and treatment. Probability values of <0.05 were considered to be statistically significant. All data are reported as least-squares means.

	RESULTS

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Animal health.

The repeated biopsies caused minimal animal discomfort. The mammary glands healed without infection and showed no evidence of clinical mastitis, and there were no long-term adverse effects on milk production or composition.

Milk production and composition.

Dietary treatment did not affect either dry matter intake or daily milk production (Table 2 ). Milk protein was increased by 0.34 percentage units (P < 0.003), resulting in higher milk protein production (P < 0.002) when the cows were fed the MFD diet. The milk fat percent and yield were reduced by 43% (P < 0.001), and the fat-corrected milk yield was decreased by 18% (P < 0.002) compared with the control.

The ACC activity in the cows fed the control diet averaged 9.8 nmol/(min · mg protein), and FAS activity was 13.2 nmol of oxidized NADPH/(min · mg protein) (Table 6 ). These activities were similar to results reported in other studies (Akers et al. 1981 , Mellenberger et al. 1973 ) for mammary tissue from lactating cows. Acetyl-CoA carboxylase and FAS activities were reduced by the MFD diet, and the effect was more pronounced for the ACC activity, which was inhibited by 62% (P < 0.001) compared with a 44% (P < 0.001) decrease in FAS activity. A parallel decrease (P < 0.001) in the mammary ACC mRNA relative abundance was observed in cows fed the MFD diet (Table 6) , and the depressions of both ACC activity and ACC mRNA relative abundance were of a similar magnitude.

	DISCUSSION

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Increased tFA formation in the rumen (Kalscheur et al. 1997a ) and higher incorporation of tFA in the milk fat (Gaynor et al. 1995 , Griinari et al. 1998 , Kalscheur et al. 1997a , Romo et al. 1996 ) are some of the changes associated with milk fat depression. Indeed reduced milk fat and increased tFA were observed in the cows fed the MFD diet in this study. When we examined the isomer distributions of milk CLA and tFA, specific changes were observed in cows fed the MFD diet. Based on the changes in the isomer profiles, we postulate that the isomerization of PUFA to conjugated dienes and the conversion of CLA to trans monoenes in the rumen were affected by the diet.

An analysis of the CLA showed a decrease in the major cis-9, trans-11 isomer and an increase in trans-10, cis-12 and trans-7, cis-9 CLA in cows fed the MFD diet. The trans-10-18:1 was the predominant trans monoene in the milk fat during milk fat depression compared with a preponderance of the trans-11-18:1 in the control cows. The changes in the cis-9, trans-11 CLA and trans-11-18:1 isomers were in agreement with the positive correlation found between these isomers in cow’s milk (Jiang et al. 1996 ). The reduction observed in cows fed the MFD diet showed that it is unlikely that these isomers are involved in reducing milk fat. Data from studies with lactating mice fed trans-11-18:1 (unpublished data) and abomasal infusion of cis-9, trans-11 CLA in lactating cows (Baumgard et al. 2000 ) are consistent with the lack of an effect of these isomers on milk fat depression.

We found 3- and 10-fold increases, respectively, in tFA and CLA isomers containing a trans-10 double bond in the milk fat of cows fed the MFD diet. The trans-10, cis-12 CLA is probably produced in the rumen via action of a specific isomerase and further hydrogenated to trans-10-18:1 (Griinari and Bauman 1999a ). The presence of trans-10, cis-12-18:2 has been reported or tentatively identified in studies in vitro with rumen fluid (Fellner et al. 1997 ) and in rumen and milk samples from cows (Griinari et al. 1997 and 1999b ). Based on infusion studies with CLA isomers, Baumgard et al. (2000) concluded that the trans-10, cis-12 CLA isomer is responsible for milk fat depression, and our findings are consistent with this hypothesis. Nevertheless, the observation of milk fat depression during the infusion of a CLA mixture containing the cis-8, trans-10, isomer (Chouinard et al. 1999b ) suggests that other CLA isomers may also be involved. Although we did not observe an increase in the cis-8, trans-10 isomer (Table 5) , we did find a significant increase in the trans-7, cis-9 CLA in the milk of cows fed the MFD diet. However, its importance in eliciting milk fat depression is unclear.

The identity of trans-7, cis-9 CLA was elucidated by Yurawecz et al. (1998) , and it has been described as the most abundant minor CLA in bovine and human milk. Formation of this isomer by the action of ⁹-desaturase on trans-7-18:1 has been reported to occur in microsomal preparations of rat liver (Pollard et al. 1980 ), and Corl et al. (1998) showed that cow mammary gland can synthesize cis-9, trans-11 CLA from trans-11-18:1. Thus it is possible that trans-7, cis-9 CLA could originate from ⁹-desaturation of trans-7–18:1 in mammary tissue. Further studies are required to determine whether this isomer is produced in the rumen.

Based on the FA composition (Table 3) in the milk of cows fed the MFD diet, the decrease in milk fat content was mostly related to a reduction in FA synthesized de novo in mammary tissue. Lower yields of these FA were consistent with observations of reduced ACC and FAS activity and ACC mRNA relative abundance. From the changes in ACC mRNA relative abundance, it is likely that the reduction in enzyme activities is due to a decreased content of these enzymes in the milk fat–depressed state.

PUFA can suppress lipogenic gene expression (Clarke and Jump 1994 , Jump and Clark 1999 , Jump et al. 1994 ). Although some dietary PUFA escapes rumen biohydrogenation, we do not believe that PUFA were directly involved in milk fat depression in this work. In previous studies (Gaynor et al. 1994 , Romo et al. 1996 ) in lactating cows postruminally infused with identical amounts of PUFA, we observed a reduction in milk fat only when the infusion mixture contained trans-FA. Loor and Herbein (1998) reported that compared with abomasal infusion of linoleic acid alone, CLA was required before milk fat depression was demonstrated. In addition, when cows were fed diets supplemented with oils rich in PUFA (Kalscheur et al. 1997b ), milk fat depression was not observed, and when cows were fed identical levels of dietary PUFA, milk fat was reduced only when rumen pH was lowered (Kalscheur et al.1997a ). These studies suggest the conclusion that dietary PUFA per se are not sufficient to induce milk fat depression and that alterations in the rumen environment are also required. Such alterations were undoubtedly responsible for the increased levels of specific isomers observed in the present study, and we believe that one or more of these isomers are involved in milk fat depression.

In summary, the feeding of an MFD diet to lactating cows decreased de novo FA synthesis in the mammary gland. Changes in mammary ACC mRNA relative abundance were consistent with decreased de novo FA synthesis, indicating that the mechanism of milk fat depression may involve, at least in part, the regulation of gene expression of lipogenic enzymes. The alterations in the CLA and trans-18:1 isomer patterns were an indication of specific modifications in the rumen environment induced by the MFD diet. The results were in agreement with an effect of the trans-10, cis-12-CLA on milk fat depression, but the changes observed in other tFA and CLA isomers may also be important.

	FOOTNOTES

 
¹ Supported in part by the Maryland Agricultural Experiment Station.

³ Abbreviations used: CLA, conjugated linoleic acid(s); MFD, milk fat–depressing; ACC, acetyl-CoA carboxylase; FA, fatty acid(s); FABE, fatty acid butyl esters; FAME, fatty acid methyl esters; FAS, fatty acid synthase; PUFA, polyunsaturated fatty acids; GLC, gas-liquid chromatography; HPLC, high performance liquid chromatography; tFA, trans-18:1 fatty acid(s).

Manuscript received March 13, 2000. Initial review completed April 8, 2000. Revision accepted June 27, 2000.

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