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© 2008 American Society for Nutrition J. Nutr. 138:449-454, March 2008

Biochemical, Molecular, and Genetic Mechanisms

Conjugated Linoleic Acid Reduces Body Fat Accretion and Lipogenic Gene Expression in Neonatal Pigs Fed Low- or High-Fat Formulas1–3,

Benjamin A. Corl, Susan A. Mathews Oliver, Xi Lin, William T. Oliver, Yongxi Ma, Robert J. Harrell and Jack Odle*

Department of Animal Science, North Carolina State University, Raleigh, NC 27695

* To whom correspondence should be addressed. E-mail: jack_odle{at}ncsu.edu.


   ABSTRACT
TOP
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
Childhood obesity is an increasing problem and may predispose children to adult obesity. Weight gain during infancy has been linked to excessive weight later in life. Conjugated linoleic acids (CLA) have been shown to reduce fat gain and body fat mass in animal models and in humans. The effects of CLA in a piglet model of human infancy have not been determined. The objective of this experiment was to examine the regulation of body composition and lipid metabolism in pigs fed low- and high-fat milk formulas supplemented with CLA. Twenty-four piglets were fed low- (3%) or high-fat (25%) diets with or without 1% CLA in a 2 x 2 factorial design. Formulas were fed for 16–17 d. Piglet body weight gains did not differ, although pigs fed the low-fat diets consumed greater amounts of diet. Piglets fed the high-fat formula accreted 50% more body fat during the feeding period than low-fat fed piglets and CLA reduced body fat accretion regardless of dietary fat content. Liver and muscle in vitro oxidation of palmitate was not influenced by dietary treatments. Adipose tissue expression of acetyl-CoA carboxylase-{alpha} and lipoprotein lipase were significantly reduced by CLA treatment. Overall, CLA reduced body fat accretion without influencing daily gain in a piglet model of human infancy. Results indicate that inhibition of fatty acid uptake and synthesis by adipose tissue, and not increased fatty acid oxidation in liver or muscle, were involved in reducing body fat gain.


   Introduction
TOP
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
Childhood obesity has increased 3-fold in the last 20 y with potentially long-lasting effects on health. Approximately 4% of children aged 6–11 y were considered obese in NHANES II (1976–1980), but the percentage increased to 13% by NHANES III (1988–1994) (1). Many factors have potentially contributed to the increase in childhood obesity, including decreases in physical activity and increases in the availability of foods high in fat and sugar (2). Evidence suggests that weight gain early in life may influence the probability of becoming overweight and obese into adulthood (3,4). Nutritional intervention during this time period may reduce the probability of developing obesity later in life.

Conjugated linoleic acids (CLA)4 are unsaturated fatty acids with conjugated diene bonds in a combination of cis and/or trans spatial configurations. Positive biological effects are associated with CLA and include anticarcinogenic properties, antiatherogenic effects, and antidiabetogenic effects, especially in animal models (5). CLA has been shown to affect lipid metabolism (6) and reduce fat mass in mice (7), pigs (8), and humans (9). Meat and dairy products from ruminants are the major source of cis-9, trans-11 CLA in human diets (10), but commercial supplements contain equal proportions of cis-9, trans-11 CLA and trans-10, cis-12 CLA (11). CLA are secreted in human milk (12) and Innis and King (13) found a positive relationship between the CLA in breast milk and in the plasma lipid of the infant.

Infant formulas are formulated to emulate human milk, but many components are not present. Fortification of infant formulas with arachidonic and docosahexaenoic acids has been approved to stimulate retinal and neuronal development and is widely accepted among consumers. The effects of CLA on infant growth and body composition have not been established. The piglet has proven to be a suitable model for comparison to the human infant when studying lipid nutrition and the piglet has many similarities with human infants, including the development of the intestine, fat digestion and absorption, and also many of the pathways of lipid metabolism (14). The objective of this study was to examine the regulation of body composition and lipid metabolism in piglets fed low- and high-fat milk formulas supplemented with CLA isomers.


   Materials and Methods
TOP
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 LITERATURE CITED
 
    General animal care. The Institutional Animal Care and Use Committee of North Carolina State University approved all procedures. A total of 24 piglets from 7 sows were obtained from the North Carolina State University Swine Educational Facility and moved to the Grinnells Intensive Swine Research Laboratory at ~1 d of age. The piglet housing and feeding system was described previously by Mathews et al. (15). Piglets were randomly assigned to 1 of the following 4 dietary treatments (Table 1): 1) high fat, containing 25% fat (HF; n = 6); 2) high fat supplemented with 1% CLA (HF+CLA; n = 6); 3) low fat, containing 3% fat (LF; n = 6); or 4) low fat supplemented with 1% CLA (LF+CLA; n = 6). All diets met or exceeded piglet requirements (16). CLA was supplied in the diet as fatty acid methyl esters. Sunflower oil (SO) methyl esters were added to HF and LF diets (1%) to match the methyl ester concentration of the CLA-containing diets (Table 2). At the end of the study (d 16 or 17), piglets were killed via American-Veterinary-Medical-Association–approved electrocution followed by exsanguination. An initial group of 10 piglets from 5 litters was used as a reference for calculation of nutrient accretion.


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  		TABLE 1 Formulated composition and calculated analysis of the LF (3%) and HF (25%) formulas with 1% SO or 1% CLA methyl esters fed to piglets1

 
    Animal feeding and diets. Diets were reconstituted at 150 g/L of water (~11 g dry matter/100 g) and the feeding system was adapted from McClead et al. (17). Formula consumed ad libitum by piglets was refreshed 4 times daily (0800, 1300, 1800, and 2300). Formula was reconstituted on a daily basis and stored at 4°C until it was fed. Co-EDTA was prepared as described by Uden et al. (18) and added to diets (0.1 g/100 g of dry diet) ~36 h prior to removal of pigs from the experiment as an inert marker of dry matter digestibility. All components of the feeding system were thoroughly cleaned with a liquid chlorinated detergent (DS Liquid, Command, Diversey) each day prior to the first feeding at 0800.

    Performance and blood collection. Pigs were weighed and formula intake was determined gravimetrically daily. Blood was collected via jugular venipuncture on d 16 or 17 of the study at 0900 after all piglets had been fed. After collection, blood samples were centrifuged (Sorvall, model 64000) at 825 x g; 10 min at 4°C. Plasma was collected and aliquots were frozen at –20°C for plasma urea nitrogen (PUN) and nonesterified fatty acid (NEFA) analyses. PUN was assayed by the quantitative urease/Berthelot procedure (Sigma Diagnostics, Sigma) and plasma NEFA concentration was analyzed by an enzymatic colorimetric method (Wako Pure Chemical Industries).

    Body composition and fatty acid analysis. The whole body was ground and thoroughly mixed (TorRey model M22-R-2) using an 82.6-mm kidney plate (TorRey model TOR 22KP) followed by a 15.9-mm plate (TorRey model TOR 12P 5/8) and then a 4.8-mm plate (TorRey model TOR 12P 3/16). Subsamples were taken, freeze-dried, powdered in liquid nitrogen, and stored at –20°C until proximate analysis. Water content was calculated by weight loss after drying at 100°C for 24 h in a forced-air oven (19). Total body crude protein was determined using the Kjeldahl procedure (19). Total fat was assayed using the Folch procedure (20) and an internal standard (C17:0) was added to each sample prior to tissue homogenization. Fatty acid methyl esters were quantitatively analyzed by GLC with flame ionization detection (21).

    In vitro β-oxidation. Immediately after exsanguination, the liver and semitendinosus muscle were removed and weighed. A section of each tissue was placed in tissue homogenate buffer (220 mmol/L mannitol, 70 mmol/L sucrose, 2 mmol/L HEPES, and 0.1 mmol/L EDTA) at a ratio of 1:7 mL. Tissues were homogenized on ice with a handheld Potter-Elvehjem homogenizer until tissue was evenly dispersed into buffer. Homogenates were cleared by centrifugation at 750 x g; 15 min and the supernatant transferred to a weighed and labeled vial. Protein concentration was determined by the Biuret method. β-Oxidation experiments using 14C-palmitate (ARC) and determination of 14C in CO2, acid soluble products, and esterified products were conducted as previously described by Odle et al. (22). Oxygen consumption was measured using the YSI Biological Oxygen Monitor (Model 5300, YSI) as previously described (22).

    Real time RT-PCR. Immediately after exsanguination, subcutaneous adipose tissue samples were collected, snap frozen in liquid nitrogen, and stored at –80°C for RNA analysis. RNA was extracted using TRI Reagent (Sigma) according to manufacturer's instructions. RNA was quantified by measuring absorbance at 260 nm and the integrity of the 18s and 28s ribosomal RNA was determined visually after electrophoresis in a 1% agarose gel stained with ethidium bromide. First-strand cDNA synthesis was completed using Omniscript Reverse Transcriptase kit (Qiagen) according to manufacturer instructions with 2 µg total RNA using oligo(dT) (Roche Applied Science) as the primer. RNase inhibitor (Roche Applied Science) was included in the reaction. Quantification of gene transcripts for acetyl-CoA carboxylase-{alpha} (ACC-{alpha}), fatty acid synthase (FAS), malic enzyme (ME), lipoprotein lipase (LPL), glycerol-3-phosphate acyltransferase (GPAT), and diacylglycerol acyltransferase (DGAT) was completed using gene-specific primers (Supplemental Table 1) by real-time RT-PCR using the Quantitect SYBR Green PCR kit (Qiagen) according to the manufacturer's instructions with 1 µL of cDNA in a 20-µL total reaction. β-Actin was used as the endogenous control and negative controls were included on each 96-well plate. Primers were designed to span introns using DNASTAR Primer Select software (23). Primers were designed using known pig sequences for all transcripts except ACC-{alpha}. Primers for ACC-{alpha} were designed using the human ACC-{alpha} transcript sequence. The identity of PCR products was confirmed by sequencing and alignment with known transcript sequences. Fluorescence measurements were recorded in real time with an OPTICON real-time thermal cycler (MJ Research). Data were analyzed using the 2–{Delta}{Delta}CT procedure (24) following verification that amplification efficiencies were similar. The cycle threshold values were calculated as the cycle number at which fluorescence of the sample exceeded threshold (threshold was determined by multiplying the SD of the baseline by 10).

    Statistical analysis. Data were analyzed as a 2 x 2 factorial, completely randomized design using the general linear models procedure of SAS (SAS Institute). Treatment differences were evaluated using the main effect of fat level (FL) and CLA, as well as the interaction. Differences were considered significant at P < 0.05.


   Results
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 LITERATURE CITED
 
    Growth, food intake, and digestibility. Over the treatment period, piglet body weight gains and body weights did not differ among the treatment groups (data not shown). Piglets fed the LF diets consumed greater amounts of formula than HF-fed piglets. However, due to the energy density of the LF diet, metabolizable energy intake was 19% less in the LF-fed piglets (P < 0.01; data not shown). CLA did not affect daily body weight gain, formula intake, or feed conversion. PUN and NEFA were increased 68 and 110%, respectively, in HF-fed piglets compared with LF-fed piglets (data not shown). CLA did not influence PUN or NEFA.

Although ileal apparent dry matter digestibility of the diets was decreased by CLA (P < 0.01), rectal apparent dry matter digestibility was not affected by FL (P > 0.10) or CLA (P > 0.50; data not shown). Ileal lipid digestibility was lower in LF-fed piglets (P < 0.01). Additionally, CLA supplementation reduced ileal lipid digestibility, with a greater reduction in the HF-fed piglets than in the LF-fed piglets (interaction, P < 0.03) (data not shown).

    Whole body tissue and fatty acid accretion rates. Although dietary treatments did not affect daily body weight gains, the composition of gain was influenced by treatments. Feeding the HF diet increased accretion of fat by 50% compared with the LF diet (Table 3). CLA reduced fat accretion regardless of dietary FL and tended to reduce total body protein accretion (P < 0.06). Dietary treatments did not affect overall whole body accretion of ash or water.


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  		TABLE 3 Total body accretion of piglets fed a LF (3%) or HF (25%) formula with 1% SO or 1% CLA methyl esters1

 

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  		TABLE 2 Analyzed fatty acid composition of the LF (3%) and HF (25%) formulas with 1% SO or 1% CLA methyl esters1

 
In addition to the influence of dietary treatments on fat accretion, fatty acid accretion also was influenced by dietary treatments. Only piglets fed CLA-containing diets had detectable whole body concentrations of CLA (Table 4). Piglets fed the HF formula accreted more medium-chain and 18-carbon fatty acids than piglets fed the LF formula, as would be expected with the increased content of these fatty acids in the HF diet (Table 2). LF-fed piglets also had reduced accretion of 22:6 compared with the HF-fed piglets. CLA reduced accretion of cis-9 desaturated fatty acids, including 14:1, 16:1, cis-18:1, and 20:1, but did not influence the accretion of 20:4 or 22:6.


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  		TABLE 4 Daily accretion of fatty acids from piglets fed a LF (3%) or HF (25%) diet with 1% SO or 1% CLA methyl esters12

 
    In vitro β-oxidation. Oxidation of palmitate by muscle and liver homogenates was measured to determine whether alterations in body fat could be attributed to changes in the oxidation of fatty acids. CLA and FL did not influence total β-oxidation of palmitate by liver or skeletal muscle (Fig. 1). Contributions of mitochondrial and peroxisomal oxidation to total β-oxidation were similar among treatments and the accumulation of radiolabel in esterified products in liver incubations did not differ (data not shown). Liver oxygen consumption rates also were unaffected by CLA or FL (data not shown).


Figure 1
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  		FIGURE 1  Total β-oxidation of 14C-palmitate in the liver and muscle of piglets fed a high (25%) or low (3%) fat formulas with 1% SO methyl esters or 1% CLA methyl esters. Values are means ± SEM, n = 6. Variables were not affected by CLA, FL, or their interaction.

 
    Gene expression. The gene expression of several enzymes involved in fatty acid and triglyceride synthesis was examined in adipose tissue. Dietary FL and CLA feeding did not influence the expression of fatty acid synthetase or GPAT (Table 5). The HF diet reduced ME expression and also tended to reduce DGAT expression. The expressions of ACC-{alpha} and LPL were reduced by CLA feeding.


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  		TABLE 5 Real-time RT-PCR data for genes of lipid metabolism in adipose tissue from piglets fed a LF (3%) or HF (25%) formula with 1% SO or 1% CLA methyl esters1

 

   Discussion
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
LITERATURE CITED
 
The prevalence of childhood obesity has doubled over the past 20 y (1). Increases in the number of overweight children may lead to future health problems in our society such as increased prevalence of diabetes and atherosclerosis. Identifying interventions to reduce the rate of obesity will improve the overall health of the obese population, thereby reducing obesity related health disorders. Early childhood weight gain may influence the development of adulthood obesity (3,4). Defining specific nutritional components that are potential modifiers of adipose expansion at critical periods in development are possible strategies to modify body fatness. Currently, both long-chain PUFA and CLA are agents involved in the modulation of lipid metabolism (6,25). To date, no studies have investigated how CLA affects the neonate, but it is an important area to investigate, because CLA is found in human milk and infant formulas (12). CLA has been shown to reduce body fatness in several animal models, including the pig (8,26,27), and may have beneficial affects in reducing the incidence of obesity.

Our primary objective was to determine the ability of CLA to reduce body fatness in growing neonates fed LF and HF diets. After 17 d, neither FL in the diet nor supplementation with 1% CLA affected overall piglet growth and piglet performance was similar to other trials conducted in our laboratory (15). Improvements in feed conversion are often observed with CLA supplementation (8,26–28), but these effects were not observed in this experiment. Body fat accretion was increased by diets with higher levels of dietary fat and was reduced by CLA. To our knowledge, this is the first report of reduced body fat accretion in neonatal animals. Reductions in body fat accretion have been observed previously with older pigs (8) as well as reductions in total body fat (26,27). Interestingly, no interaction of CLA with dietary fat content was observed, demonstrating CLA's ability to modulate both de novo fatty acid synthesis as well as deposition of fatty acids from circulation.

A secondary objective of this work was to explain more mechanistically how CLA reduces body fat accretion in these pigs. Several explanations have been proposed based on results from different animal models. Reductions in intake (27) and diet digestibility have been reported (29). In this study, we did not find an effect of CLA on feed intake, but CLA reduced the ileal apparent dry matter digestibility of the diet. In mice, the reduced diet digestibility accounted for 26% of the loss in total body energy (29). In this experiment, reductions in diet digestibility were small and accounted for a minor portion of reduced body fatness.

Reductions in body fat without a difference in intake or body weight may be caused by increased energy expenditure. The increased energy expenditure might be the result of increased basal metabolic rate, increases due to the thermic effect of absorption, digestion, and assimilation of nutrients after a meal, or an increase in physical activity. Terpstra et al. (29) observed increased energy expenditure in mice that accounted for 74% of the reductions in body energy storage. Others also have observed similar increases in energy expenditure (30,31).

In this study, we measured in vitro β-oxidation of palmitate in liver and muscle. CLA has been shown to be a potent activator for the PPAR{alpha}, which up regulates β-oxidation (32). Others have previously reported increased fatty acid oxidation as a result of fatty acid supplementation (33–35). In this experiment, in vitro β-oxidation of palmitate was not altered by dietary CLA supplementation in liver or skeletal muscle. Also, analysis of liver tissue fatty acid composition revealed no accumulation of CLA in the liver (data not presented). However, we did find an increase in liver weights as a percentage of piglet body weight (9% increase; data not shown), similar to previously published data in rodents (31,36). PUN concentrations were higher in the HF-fed pigs, suggesting that dietary amino acids were not utilized as efficiently for protein deposition in these treatments, but CLA did not influence PUN, indicating that amino acid oxidation was not altered by this treatment. Furthermore, data from PPAR{alpha}-null mice indicates that although CLA is a potent activator of PPAR{alpha}, the effects of CLA on body composition are independent of this nuclear receptor (37).

Changes in fatty acid accretion were evident as a result of CLA supplementation and agreed with previous observations. Accretion of individual fatty acids was not affected by an interaction between dietary fat content and CLA. The most striking effect of CLA on fatty acid accretion was the reduction in cis-9 monounsaturated fatty acids. Reductions in cis-9 monounsaturated fatty acids and stearoyl-CoA desaturase activity have previously been observed (28,38). Dietary CLA reduces liver stearoyl-CoA desaturase mRNA (39) and can directly inhibit activity (40,41). Resulting reciprocal increases in adipose SFA content have been shown to improve pig carcass quality due to increased belly firmness, an important processing characteristic (28,38). Changes in stearoyl-CoA desaturase activity are associated with reductions in body fatness. Specifically, stearoyl-CoA desaturase-null mice have greatly reduced body fatness and evidence suggests that liver activity of this enzyme is crucial for normal lipid metabolism (42). Although the effects of CLA on stearoyl-CoA desaturase and monounsaturated fatty acids are consistent and correlate well with reductions in body fat accretion, evidence from stearoyl-CoA desaturase-null mice indicates that changes in stearoyl-CoA desaturase activity are not required for reductions in body fat (43).

Another mechanism for CLA to control adipose tissue lipid filling is through reductions in the gene expression of enzymes for fatty acid synthesis, uptake, and triglyceride synthesis. In this study, we utilized piglets that synthesize lipids in adipose tissue, whereas humans synthesize large quantities of fatty acids in the liver. Although species differences in lipogenic tissue do exist, similar mechanisms for the regulation of lipid synthesis exist in these tissues and species that utilize liver, adipose, or both tissues have been shown to respond to CLA supplementation (7–9,44,45). As with other measurements, dietary fat content did not influence the effects of CLA on gene expression of measured transcripts in adipose tissue. Feeding CLA reduced mRNA abundance of ACC-{alpha} and LPL. LPL hydrolyzes extracellular triglyceride to enable subsequent cellular uptake of released FFA. Reduced LPL gene expression and activity in adipocytes is a well-characterized effect of CLA (46,47). ACC-{alpha} is the rate-limiting enzyme for de novo fatty acid synthesis and reductions in gene expression have been observed in hamsters (46), dairy cows (48), and mice (49). Reductions in lipogenic activity are typically associated with reductions in ACC-{alpha}, ME, and FAS; however, FAS was not reduced in this study. Transcriptional control of these genes in adipose tissue is mediated by sterol regulatory element-binding protein-1c and PPAR{gamma}. Reductions in the mature, DNA-binding portion of SREBP-1 have been observed in bovine mammary epithelial cells (50), but evidence for this mechanism in adipose tissue or cultured adipocytes is not documented. Regulation of PPAR{gamma} by CLA has been demonstrated in culture human adipocytes (51). Modulation of PPAR{gamma} activity in cultured human adipocytes by CLA has been shown to be a result of increased production of inflammatory cytokines leading to the phosphorylation of transcription factors, possibly including PPAR{gamma} (52). Increased production of inflammatory cytokines by adipose tissue also has been observed in mice (53).

In conclusion, the results of this study suggest that CLA is a modulator of body lipid in the neonatal period that may potentially reduce the risk of obesity not only in childhood but also subsequently in adulthood. Reductions in body fat accretion were not a result of changes in diet digestibility or increases in fatty acid oxidation. Changes in the accretion of individual fatty acids were observed, especially for monounsaturated fatty acids, but we do not think alterations in the activity of stearoyl-CoA desaturase contributed to reductions in body fat accretion. The reductions in the mRNA abundance of ACC-{alpha} and LPL indicated transcriptional regulation of these key enzymes for de novo fatty acid synthesis and uptake of fatty acids from circulation by CLA were responsible for reductions in body fat accretion and were possibly mediated by changes in the activity of PPAR{gamma}.


   FOOTNOTES
 
1 Supported by the Cooperative State Research, Education, and Extension Service, USDA National Research Initiative (grant no. 2007-35206-17897 to X.L. and J.O.). Back

2 Author disclosures: B. A. Corl, S. A. Mathews Oliver, X. Lin, W. T. Oliver, Y. Ma, R. J. Harrell, and J. Odle, no conflicts of interest. Back

3 Supplemental Table 1 is available with the online posting of this paper at jn.nutrition.org. Back

4 Abbreviations used: ACC-{alpha}, acetyl-CoA carboxylase-{alpha}; CLA, conjugated linoleic acid; DGAT, diacylglycerol acyltransferase; FAS, fatty acid synthase; FL, fat level; GPAT, glycerol-3-phosphate acyltransferase; HF, high fat; LF, low fat; LPL, lipoprotein lipase; ME, malic enzyme; NEFA, nonesterified fatty acids; PUN, plasma urea nitrogen; SO, sunflower oil. Back

Manuscript received 17 September 2007. Initial review completed 23 October 2007. Revision accepted 11 December 2007.


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 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
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