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Nutrient-Gene Interactions

Trans10,cis12–18:2 Is a More Potent Inhibitor of De Novo Fatty Acid Synthesis and Desaturation than cis9,trans11–18:2 in the Mammary Gland of Lactating Mice¹

Dairy Science Department, Virginia Tech, Blacksburg, VA 24061-0315

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
To investigate the effects of 2 conjugated linoleic acid (CLA) isomers and trans11–18:1 (TVA) on de novo lipogenesis and desaturation in liver and mammary gland, lactating mice were fed diets containing 3% canola oil (control) or 2% canola oil plus 1% stearic acid (SA), TVA, cis9,trans11 CLA (c9t11), or trans10,cis12 CLA (t10c12). In mammary tissue, TVA and CLA isomers reduced mRNA for acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) compared with control, but only c9t11 and t10c12 reduced mammary ACC activity. Of the 2 CLA isomers, t10c12 caused a greater reduction in mammary ACC activity. Hepatic ACC or FAS activity and mRNA abundance were not affected by dietary treatments. Feeding TVA, c9t11, or t10c12 reduced mammary stearoyl-CoA desaturase 1 (SCD) mRNA and activity. Reduction was greater due to feeding t10c12 compared with c9t11. Hepatic SCD mRNA was not affected by dietary treatments, but both CLA isomers depressed hepatic SCD activity. Results indicated that t10c12 is a more potent inhibitor of mammary lipogenesis and desaturation than is c9t11. A net gain of 77 and 1690 µg of c9t11 in liver and mammary tissue, respectively, was found in the TVA-fed group over the control and SA-fed group. However, reduced mammary SCD mRNA or activity due to feeding TVA may indicate a limited capacity for desaturation of dietary TVA to c9t11 in vivo.

KEY WORDS: • stearoyl-CoA desaturase • conjugated linoleic acid • trans-vaccenic acid • lipogenesis • mammary gland

Extensive research indicates that conjugated linoleic acids (CLA)⁴ may confer many beneficial health effects on humans (1). Feeding CLA mixtures reduced body fat and increased lean body mass in laboratory animals (2–4). Results from feeding CLA mixtures enriched with trans10,cis12 CLA (t10c12) suggested that this isomer is a potent antilipogenic agent (5,6). However, conflicting results have been reported on the effects of CLA on lipid and glucose metabolism. Ryder et al. (7) demonstrated improved insulin sensitivity and glucose tolerance in Zucker diabetic fa/fa rats from feeding a CLA mixture [50:50, t10c12 and cis9,trans11 CLA (c9t11)], but not c9t11. In contrast, a CLA-enriched diet induced insulin resistance and hyperlipidemia in C57BL/6J mice (8). Pure t10c12 also induced hyperinsulinemia in mice (9).

When fed to growing mice, mixtures of CLA (10) or t10c12 (9) induced liver enlargement due to lipid accumulation. One of the mechanisms of lipid accumulation in response to CLA feeding may involve upregulation of lipogenic enzyme genes such as fatty acid synthase (FAS), spot 14, ATP-citrate lyase, malic enzyme, and stearoyl-CoA desaturase 1 (SCD) (11). Induction may be due to upregulation of sterol regulatory element-binding protein 1 (SREBP-1) (9,11), the transcriptional factor activating transcription of lipogenic enzyme genes (12,13).

In direct contrast to the stimulatory effect of CLA isomers on hepatic SCD mRNA abundance, it was demonstrated that t10c12, but not c9t11, inhibited SCD mRNA expression in the liver of growing mice in vivo (14) or hepatic SCD activity in vitro (15). The c9t11 isomer, but not t10c12, reduced hepatic SREBP-1c mRNA and its active form in ob/ob mice, a model of obesity, hyperlipidemia, and insulin resistance (16). The mRNA abundance of hepatic acetyl-CoA carboxylase (ACC), FAS, or SCD, target genes of SREBP-1c, was not quantified in that study. Nondiabetic wild-type mice also were not included.

During lactation, the rate of fatty acid synthesis in mouse liver increases (17) and may contribute to the fatty acid supply for the mammary gland. Desaturation of stearic acid by hepatic SCD may be an important source of oleic acid for secretion into milk fat by the mammary gland (18). However, it is not known whether and how CLA isomers affect hepatic lipogenesis or desaturation in lactating mice.

Abomasal infusions of CLA mixtures reduced concentrations and yields of SCFA and medium-chain fatty acids (MCFA), the products of de novo lipogenesis (19). Abomasal infusion of pure t10c12 resulted in lower milk fat concentration and yield (20,21) along with reduced mRNA abundance of ACC, FAS, and SCD in bovine mammary tissue (22). Enrichment of c9t11 and t10c12 in milk fat was observed when lactating women consumed a CLA supplement (60% total CLA), but their milk fat concentration was reduced by 23% (23). Whether similar molecular mechanisms are responsible for the observed milk fat depression in human mammary tissue remains to be determined. Lactating mice may be a useful model for such evaluation (24).

Rodent hepatocytes have the capacity for desaturation of exogenous trans-vaccenic acid (TVA), the primary trans-18:1 in bovine milk fat, to c9t11 via SCD (25). Desaturation of TVA may be responsible for a portion of the elevated concentration of c9t11 in human milk when lactating women consume dairy products (26). Consumption of a diet enriched with TVA increased the concentration of c9t11 in human serum (27). In addition, TVA was converted to c9t11 in human mammary and colon cell lines in a dose-dependent fashion (28).

In the present study, the effects of dietary TVA and purified CLA isomers (c9t11 vs. t10c12) on lipogenesis and desaturation in liver and mammary gland of lactating mice were evaluated. We determined enzymatic activity and mRNA abundance for ACC and FAS, important lipogenic enzymes in de novo fatty acid synthesis (29,30). Activity and mRNA for SCD also were determined to evaluate the capacity for desaturation of TVA to c9t11 (31,32).

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Animals, diets, and design. Details about the experimental design were presented previously (33). Briefly, pregnant mice (n = 28; Harlan-Teklad) were maintained in a temperature-controlled room (23°C) with a 12-h light:dark cycle and fed the breeder diet (Harlan-Teklad mouse breeder diet, S-2335) with water available at all times. After review and approval by the Virginia Tech Animal Care Committee, the experimental protocol was carried out in accordance with the NIH guidelines (34).

The breeder diet (970 g) was supplemented with 30 g canola oil (64% oleic acid, Hunt-Wesson) (control) or 20 g canola oil plus 10 g stearic acid (SA) (N-18-A, 99% purity, Nu-Chek-Prep), 10 g TVA (U-49-A, 99% purity, Nu-Chek-Prep), 10 g c9t11 (Natural Lipids), or 10 g t10c12 (Natural Lipids) per kg diet. The c9t11 contained 90.7% cis9,trans11–18:2 and 1.0% trans10,cis12–18:2, and t10c12 contained 96.2% trans10,cis12–18:2 and 1.8% cis9,trans11–18:2. Dietary groups are designated control, SA, TVA, c9t11, and t10c12 in tables and figures. The breeder diet contained 17.5, 11.7, and 53.7% protein, fat, and carbohydrate, respectively. The dietary fatty acid composition was described (33).

Mice were randomly assigned on d 4 postpartum to obtain groups of 5 (control and SA groups) or 6 (TVA, c9t11, and t10c12 groups). On the same day, the litters were reduced to 8 pups each. Two dams in the t10c12 group were removed during the early experimental phase because they reduced voluntary food intake to <5 g/d. On d 15, dams were killed by cervical dislocation. Liver and mammary glands were excised, weighed, placed in liquid nitrogen, and stored at –80°C until subsequent analyses.

    Fatty acid analysis. Lipids were extracted from liver and mammary tissue with chloroform/methanol (2:1, v:v) (35). Fatty acids in tissue lipids were quantified as previously described (33).

    Preparation of cytosolic and microsomal fractions and enzymatic activity assay. Microsomal fractions for SCD activity were obtained using the protocol of St John et al. (36). The supernatant was kept as the cytosolic fraction and was used for determining ACC and FAS activity. The protein content of the 2 fractions was determined using the spectrophotometric bicinchoninic acid protocol (Pierce). The procedures for ACC, FAS, and SCD activity were based on those reported by Gregolin et al. (37), Smith and Abraham (38), and St John et al. (36), respectively. Specific activities are reported as the amount of product formed per minute per milligram of protein.

    cDNA probes. Plasmids pKK160, pBR322, and pGEM-7Zf⁺ contained cDNA for the mouse SCD, rat FAS, and sheep ACC, respectively. Insert cDNA was excised from plasmids and labeled using -³²P[dATP] and a random priming kit (Promega).

    Northern blot analysis. Total RNA from liver and mammary tissue was extracted using TRI Reagent (MRC), following the manufacturer’s protocol. Total RNA of mammary gland (30 µg) and liver (20 µg) was separated by electrophoresis on a 0.8% agarose-formaldehyde gel, and then transferred and immobilized onto a Magna Charge nylon membrane (MSI) (39).

Prehybridization and hybridization were conducted according to the standard protocol with some modifications (40). Briefly, prehybridization was conducted for 30 min at 65°C. Hybridization was performed for 16 h with a ³²P-labeled cDNA probe (5 µg/L) specific for ACC (at 42°C), FAS (42°C), or SCD (65°C). After hybridization, membranes were washed under high-stringency (for SCD probe) or medium-stringency conditions (for ACC or FAS).

The binding of the labeled probe to target mRNA was detected by autoradiography of the hybridized membrane on Kodak X-Omat film with 2 intensifying screens at –80°C. The densities of bands for ACC, FAS, SCD, and 18S rRNA were scanned on a laser densitometer. The density of each mRNA band was normalized to that of 18S rRNA. The mRNA abundance for treatment groups was expressed relative to that of the control group (100%).

    Statistical analyses. Data are reported as least squares means ± SE. All data were analyzed using the MIXED procedure of SAS (41). Differences between treatment means were established using the Tukey-Kramer adjustment for multiple comparisons when the F-value was significant. Means were considered significantly different if P < 0.05.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Fatty acid profiles in mammary gland and liver of lactating mice. Tables 1, and 2 list the concentrations of individual fatty acids except TVA and CLA isomers in mammary tissue and liver, respectively. However, total fatty acids in each group still included treatment fatty acids. Although t10c12 did not reduce total fatty acid concentration in mammary tissue, the absolute amount of total fatty acids was lower due to feeding t10c12 (0.526 g) compared with other treatments (0.615 g). Both CLA isomers lowered the percentage of MCFA (12:0 + 14:0 + 16:0) in mammary tissue compared with control or SA (Table 1). CLA isomers also significantly lowered milk fat percentage and the percentage of MCFA in milk fat compared with other treatments, but dietary t10c12 decreased MCFA and milk fat percentage to a greater extent than c9t11 (33).

    Concentrations of TVA and CLA isomers in mammary gland and liver. Concentrations of TVA, c9t11, and t10c12 in mammary gland and liver of mice were significantly higher in the respective treatment groups (Fig. 1). The concentration of c9t11 due to feeding TVA was significantly higher in both mammary and liver tissues compared with feeding control or SA (Fig. 1B). Feeding TVA also significantly elevated c9t11 in milk fat (33). Enrichment of c9t11 due to feeding TVA also was noted in the carcass of lactating mice, as well as the liver and carcass of the pups nursing from TVA-fed dams (33).

View larger version (19K): [in this window] [in a new window]   FIGURE 1 Concentrations of TVA (A), c9t11 (B) and t10c12 (C) in mammary tissue and liver of lactating mice fed diets containing 3% canola oil (control; n = 5) or 2% canola oil plus 1% SA (n = 5), TVA (n = 6), c9t11 (n = 6), or t10c12 (n = 4). The concentrations (g/100 g total fatty acids) were quantified as described in Materials and Methods. Values are means ± SE. Means that do not share a common letter differ, P < 0.05.

View larger version (37K): [in this window] [in a new window]   FIGURE 2 Mammary mRNA abundance (A) and activity (B) of ACC and FAS of lactating mice fed diets containing 3% canola oil (control; n = 5) or 2% canola oil plus 1% SA (n = 5), TVA (n = 6), c9t11 (n = 6), or t10c12 (n = 4). Total RNA isolated from mammary tissue was used in Northern blot analysis for ACC and FAS. The radioactive hybridization signals were normalized to 18S rRNA (A). Enzymatic activities for ACC and FAS were determined as described in Materials and Methods (B). Values are means ± SE. Means that do not share a common letter differ, P < 0.05.

Mammary SCD mRNA abundance was decreased by feeding TVA (32%), c9t11 (76%), and t10c12 (27%) compared with the control (100%) (Fig. 3A). Although SA increased SCD mRNA compared with the control, SA did not alter SCD activity in mammary tissue (Fig. 3B). Both CLA isomers, however, decreased SCD activity in mammary tissue. Similar to changes in SCD mRNA, t10c12 caused a greater reduction in mammary SCD activity.

View larger version (20K): [in this window] [in a new window]   FIGURE 3 Mammary mRNA abundance (A) and activity (B) of SCD in lactating mice fed diets containing 3% canola oil (control; n = 5) or 2% canola oil plus 1% SA (n = 5), TVA (n = 6), c9t11 (n = 6), or t10c12 (n = 4). Total RNA isolated from mammary tissues was used in Northern blot analysis for SCD. The radioactive hybridization signals were normalized to 18S rRNA (A). Enzymatic activities for SCD were determined as described in Materials and Methods (B). Values are means ± SE. Means that do not share a common letter differ, P < 0.05.

View larger version (36K): [in this window] [in a new window]   FIGURE 4 Hepatic mRNA abundance (A) and activity (B) of ACC and FAS in lactating mice fed diets containing 3% canola oil (control; n = 5) or 2% canola oil plus 1% SA (n = 5), TVA (n = 6), c9t11 (n = 6), or t10c12 (n = 4). Hepatic mRNA abundance (A) for ACC and FAS was quantified as described in Figure 2. Hepatic enzymatic activities for ACC and FAS (B) were determined as described in Figure 2. Values are means ± SE.

View larger version (25K): [in this window] [in a new window]   FIGURE 5 Hepatic mRNA abundance (A) and activity (B) of SCD in lactating mice fed diets containing 3% canola oil (control; n = 5) or 2% canola oil plus 1% SA (n = 5), TVA (n = 6), c9t11 (n = 6), or t10c12 (n = 4). Hepatic mRNA abundance (A) for SCD was quantified as described in Figure 3. Hepatic enzymatic activities for SCD (B) were determined as described in Figure 3. Values are means ± SE. Means that do not share a common letter differ, P < 0.05.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Inhibition of de novo lipogenesis in the mammary gland by the CLA isomers was demonstrated by a reduction in ACC mRNA, the critical enzyme that catalyzes the committed step in de novo synthesis of fatty acids (30). Baumgard et al. (22) demonstrated reduced ACC mRNA due to exogenous t10c12 in dairy cows, but c9t11 was not included as a treatment in their study. We found a pronounced reduction in enzymatic activity of ACC by both isomers. Reduced MCFA in milk fat (33), which are solely synthesized de novo in the mammary tissue (24), provide additional evidence for the decrease in lipogenic capacity of mammary tissue in response to feeding CLA isomers. MCFA in mammary tissue also was reduced due to feeding CLA isomers. Thus, a lower milk fat percentage and MCFA in milk fat (33) were attributed to the inhibitory effects of CLA isomers on mammary lipogenesis. Greater reductions in mammary ACC activity, MCFA, and milk fat percentage due to feeding t10c12 further indicated that it was more potent than c9t11 in inhibiting mammary lipogenesis.

The inhibition of desaturation in the mammary gland was demonstrated by measuring enzymatic activity and mRNA of SCD, the enzyme catalyzing the critical step in fatty acid desaturation (31,32). Although both CLA isomers reduced mammary SCD enzymatic activity and mRNA, t10c12 caused a greater reduction. The ratio of cis9–16:1/16:0 or cis9–18:1/18:0 was significantly lower in the carcass of dams fed t10c12 (33). Similarly, feeding t10c12 resulted in a greater SA concentration in milk fat compared with other diets (33). Thus, t10c12 appeared to be a more potent inhibitor of mammary desaturation. Our results are consistent with those observed in the cows in which abomasal infusion of c9t11 as well as t10c12 caused significantly lower ratios of cis9–14:1/14:0 and cis9–18:1/18:0 in milk fat than in controls (20). Those ratios were significantly lower for t10c12 than c9t11, indicating that t10c12 was more potent than c9t11 in inhibiting SCD activity.

SREBPs, synthesized as precursors bound to the endoplasmic reticulum and nuclear envelope, are released into the nucleus upon activation (44). Three SREBP isoforms, SREBP-1a, SREBP-1c, and SREBP-2, were identified and characterized (13). SREBP-1a and -1c primarily regulate lipogenesis (44). Massive liver enlargement and triacylglycerol accumulation by feeding mixtures of CLA or t10c12 in mice were accompanied by upregulation of lipogenic enzyme genes such as FAS and SCD, mediated by elevated hepatic SREBP-1 expression that may be secondary to hyperinsulinemia due to CLA supplementation (9,11). In contrast, feeding a CLA mixture (0.5% of the diet) reduced hepatic SCD mRNA expression (14). Compared with a mixture enriched with the c9t11 isomer, only the CLA mixture containing equal amounts of c9t11 and t10c12 depressed SCD mRNA abundance, suggesting that t10c12 was responsible for the depression of SCD mRNA seen in vivo (14). The c9t11 isomer reduced hepatic SREBP-1c mRNA and its nuclear active form in ob/ob mice (16). In the present study, CLA isomers did not affect hepatic ACC or FAS mRNA or activity, but they inhibited SCD enzyme activity without affecting SCD mRNA expression in the liver. Concentrations of c9t11 and t10c12 in the mammary gland were 4.2 and 11.9 times, respectively, those in the liver when fed to lactating mice. This may explain why the mammary gland is more responsive than the liver to dietary fatty acid supplementation. To our knowledge, it is not known whether SREBP-1 is expressed in mammary gland and what role(s) it may play in lipogenesis, desaturation, or fatty acid uptake during lactation. It is also important to study a possible role for SREBP-1 in the regulation of ACC, FAS, and SCD in the mammary tissue during lactation.

Our initial in vivo study demonstrated that dietary TVA was converted to c9t11 in limit-fed lactating mice (45). We have, in the present study, confirmed and extended this observation under conditions of ad libitum consumption. The c9t11 isomer derived from dietary TVA was available for secretion into milk, and both dietary and endogenous c9t11 were readily transferred to suckling pups via milk (33). Concentrations of c9t11 were significantly higher in mammary gland and liver due to feeding TVA compared with other treatments (Fig. 1). Similarly, concentrations of TVA and c9t11 were elevated in carcass and liver of pups nursing from TVA-fed dams (33). Conversion most likely took place in the liver or mammary gland rather than adipose tissue as suggested for growing mice (46). In humans, 19% of TVA was converted to c9t11 for a TVA intake ranging from 1.5 to 4.5 g/d (47). In the present study, a net gain of 77 and 1690 µg of c9t11 in liver and mammary tissue, respectively, in the TVA-fed group over the control and SA groups was noted.

A close linear relationship between TVA and c9t11 was observed for bovine milk fat in a number of studies and across a wide range of diets [for review, see (48)], suggesting no inhibitory effect of TVA on bovine mammary SCD in vivo. We speculated that the inhibitory effect of dietary TVA on mouse mammary SCD mRNA or activity may be due to TVA itself or to c9t11 converted from TVA. This issue warrants further investigation in lactating nonruminants.

In conclusion, results from the present study indicated that CLA isomers reduce lipogenesis in the mammary gland of lactating mice, presumably by reducing enzyme activity and mRNA abundance of ACC, the critical enzyme in de novo fatty acid synthesis. CLA isomers also inhibited mammary desaturation by reducing mammary SCD activity and mRNA abundance. The t10c12 isomer of CLA appeared to be a more potent inhibitor of lipogenesis as well as desaturation in the mammary gland. Although consumption of dairy fats with TVA may result in greater c9t11 in human milk and tissues, the capacity to desaturate TVA to c9t11 may be limited due to reduced mammary SCD activity or mRNA as a result of elevated dietary TVA intake.

	ACKNOWLEDGMENTS

 
We thank R. E. Pearson for assistance with statistical analyses. The cDNA probes specific for ACC, FAS, and SCD were kindly provided by M. Barber and M. Travers (Hannah Research Institute, Ayr, Scotland), M. Magnuson (Vanderbilt University, Nashville, TN), and J. M. Ntambi (University of Wisconsin, Madison, WI), respectively.

	FOOTNOTES

 
¹ Supported by the John Lee Pratt Nutrition Fellowship Program administered through the College of Agriculture and Life Sciences of Virginia Tech.

² Present address: Lipid Research, Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110. To whom correspondence should be addressed. E-mail: xlin{at}im.wustl.edu.

³ Present address: Department of Animal Sciences, University of Illinois, 206 ERML, Urbana, IL 61801.

⁴ Abbreviations used: ACC, acetyl-CoA carboxylase; CLA, conjugated linoleic acid; c9t11, cis9,trans11 CLA; FAS, fatty acid synthase; MCFA, medium-chain fatty acids; SA, stearic acid; SCD, stearoyl-CoA desaturase; SREBP-1, sterol regulatory element-binding protein 1; TVA, trans-vaccenic acid; t10c12, trans10,cis12 CLA.

Manuscript received 13 November 2003. Initial review completed 5 January 2004. Revision accepted 18 February 2004.

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TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

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