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Article

Conjugated Linoleic Acid Persistently Increases Total Energy Expenditure in AKR/J Mice without Increasing Uncoupling Protein Gene Expression¹

David B. West^*,2, Fawn Y. Blohm, Alycia A. Truett and James P. DeLany

^* Parke-Davis Laboratory for Molecular Genetics, Alameda, CA 96501 and Pennington Biomedical Research Center, Louisiana State University, Baton Rouge, LA 70808

²To whom correspondence should be addressed.

	ABSTRACT

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AKR/J mice fed a high fat diet were treated with a 1% (1 g/100 g) admixture of conjugated linoleic acids (CLA) for 5 wk and compared with control mice. Body weights, energy intakes and energy expenditure (EE) determined by indirect calorimetry were measured weekly. CLA treatment reduced adipose depot weights by 50% but had no significant effects on either body weight or energy intake. CLA increased EE persistently by an average of 7.7% throughout the 5-wk experiment. This greater EE, despite no difference in energy intake, was sufficient to account for the lower body fat stores in the CLA-treated mice. De novo fatty acid biosynthesis in adipose tissue, measured by incorporation of deuterium-labeled water, was not decreased by CLA treatment and therefore did not explain the lower adipose lipid in these mice. Expression of uncoupling protein (UCP) in skeletal muscle, white adipose tissue and kidney was not affected by CLA treatment. In brown adipose tissue, UCP1 expression was not affected by CLA treatment. However, UCP2 expression, although quite low, was significantly greater in CLA-fed mice. We conclude that CLA acts to reduce body fat stores by chronically increasing metabolic rate. This effect on metabolic rate is likely not due to increased UCP gene expression. Furthermore, the reduced body fat is not due to decreased de novo fatty acid synthesis in white adipose tissue.

KEY WORDS: • mice • adipose tissue • metabolic rate • uncoupling protein • growth • obesity treatment

	INTRODUCTION

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Conjugated linoleic acids (CLA)³ are a group of dienoic positional and geometric isomers of linoleic acid found predominantly in foods originating from ruminants (Chin et al. 1992 and 1994 ). Even though most of the research studying the effects of CLA has used synthetically prepared CLA with a mixture of isomers, there are several indications that different isoforms may have different biological actions. For example, the cis-9/trans-11 form is derived naturally through bioisomerization of linoleic acid by rumen bacteria, and there is evidence of preferential incorporation of this form into membrane phospholipids (Ip et al. 1994 , Kepler et al. 1966 ). The cis-9/trans-11 isomer may be the active form, alone or in combination with other isomers, for the reported effects of the mixed isomer preparations on tumorigenesis in animal models (Pariza et al. 1999 ). Similarly, the trans-10/cis-12 isomer may be the active isomer affecting energy metabolism and body fat content (Park et al. 1999 )

CLA has received considerable attention due to its reported ability to inhibit carcinogenesis and to attenuate atherosclerosis in animal models. Both of these actions may be attributable to the antioxidant actions of CLA. These biological activities of CLA have been reported in several experimental models including mouse forestomach neoplasia, mouse skin carcinogenesis, rat mammary tumorigenesis, and rabbit and hamster atherosclerosis (Cook et al. 1993 , Ha et al. 1990 , Ip et al. 1991 and 1996 , Lee et al. 1994 , Miller et al. 1994 , Nicolosi et al. 1993 ).

These isomers of linoleic acid also may have effects on energy metabolism and lipid storage that could indirectly influence tumorigenesis and inflammatory responses. For example, CLA has been shown to reduce body fat and increase lean muscle in mice fed CLA in a dietary admixture (Pariza et al. 1996 , Park et al. 1997 ). Additional data from this laboratory have shown that CLA increases energy expenditure (EE) and rapidly reduces adiposity in AKR/J mice (DeLany et al. 1999 , West et al. 1998 ). Although the precise mechanism for this action of CLA is not known, the mobilization and loss of body fat stores certainly do not result from a reduction in energy consumption. We have shown in a carefully controlled study that CLA will reduce body fat stores without any reduction of energy intake (DeLany et al. 1999 ). Our intention here is to further characterize the effect of CLA on EE and attempt to elucidate or eliminate possible mechanisms. We examined the time course of the effect of CLA on total EE and determined whether the increased EE was attributable to increased expression of uncoupling protein genes. Further, we determined whether CLA treatment decreased de novo fatty acid biosynthesis in adipose tissue as one mechanism by which CLA could reduce body lipid stores. Finally, we determined whether CLA affected circulating growth hormone, another mechanism by which this fatty acid could reduce body fat content and also increase lean tissue mass.

	MATERIALS AND METHODS

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Animal husbandry and experimental design.

All studies were completed under the supervision of the Institutional Animal Care and Use Committee and the care and treatment of the mice conformed to all Public Health Service Guidelines. AKR/J male mice (4 wk old) were obtained from the Jackson Laboratory (Bar Harbor, ME). The AKR/J mouse is a useful model for studying dietary obesity and chemical agents that influence lipid mobilization from adipose tissue because this strain is particularly sensitive to dietary obesity (West et al. 1992 ). In addition, these mice share many of the metabolic characteristics of human obesity after high fat feeding (West et al. 1996 ). All mice were housed individually in hanging stainless steel cages with water bottles in a room maintained at 26 ± 2°C with a 12-h light:dark cycle.

Initially, mice consumed ad libitum Purina Mouse Chow (Diet #5015, Ralston Purina). Before treatment with dietary CLA, mice were switched to a high fat diet (Research Diets, New Brunswick, NJ, Diet #D12290) for an adaptation period of 10 d. The composition of the diet has been described previously (West et al. 1995 ). This diet contained 45, 10 and 35 g/100 g from fat, protein and carbohydrate, respectively. The fat sources were corn oil and hydrogenated coconut oil mixed to give a fatty acid composition of 1:3, saturated:unsaturated. The protein source was primarily casein with 0.4 g/4.18 kJ of DL-methionine. Carbohydrate was provided as a mixture of sucrose, maltodextrin 10 and cornstarch. The fiber source was cellulose, and mineral and vitamins were added according to the AIN76A standard (AIN 1977 ).

An adaptation period was used to minimize the effect of the transient hyperphagia observed when mice are initially given access to this high fat diet. After adaptation to the diet, mice either continued to consume the high fat diet (n = 8) or were fed a high fat diet containing 1 g/100 g CLA (n = 8). This dose of CLA was based on evidence from our previous dose-response study that 1% CLA did not decrease energy intake but still reduced carcass lipid stores. CLA was obtained from Nu-Chek-Prep (Elysian, MN) and had the following reported composition: 39.1% c9,t11 and t9,c11 CLA; 40.7% t10,c12 CLA; 1.8% c9,c11 CLA; 1.3% c10,c12 CLA; 1.9% t9,t11 and t10,t12 CLA; 1.1% c9,c12 linoleic acid; and 4.1% remainder. This composition was confirmed independently by gas chromatography. Methyl esters were prepared using sodium methoxide and then injected onto an SP2340 (Supelco, Bellefonte, PA) 100-m column at 207 kPa head pressure using a temperature program to separate the various CLA isomers.

Diets were formulated and prepared by Research Diets. In the experimental diets, CLA was substituted for corn oil. Mice were provided free access to preweighed defined diets in pellet form placed on the bottom of each cage. The mice and remaining diet were weighed two times per week and fresh preweighed diet was given. Food intake was corrected for spillage and all measurements were to the nearest 0.1 g.

Measurement of energy expenditure.

The system used for the assessment of metabolic rate in mice has been described previously (West et al. 1998 ). The four Plexiglas chambers have a floor area of 205 cm² and a height of 9.5 cm. Room air is drawn through each chamber at a rate of 0.23 L/min and then dried in a column of drierite before entering the O₂ analyzer (Model S-3A, Ametek, Pittsburgh, PA) and the CO₂ analyzer (Model CD-3A, Ametek). Each chamber was sampled once every 5 m for 75 s; only the last 45 s of the sampling period were used for analysis. During the 10 d of high fat diet adaptation, the 16 male 6-wk-old AKR/J mice were habituated to the metabolic chamber conditions by placing each mouse in a metabolic cage for one 24-h period without data collection. At the end of the dietary habituation period, mice were placed into the metabolic chambers for the measurement of a baseline EE before CLA treatment. Once a baseline was established, the mice were randomly placed in a control group (no CLA) or a treatment group (1% CLA) with 8 mice in each group. Metabolic rate measurements [EE and respiratory quotient (RQ)] were made for each mouse once per week for 5 wk. Mice were assigned randomly to the chambers each week to reduce the possibility of a chamber effect influencing the outcome. Food intake corrected for spillage was measured while the mice were in the metabolic chambers.

Measurement of lipid synthesis.

Mice were given drinking water containing 10% deuterum oxide for the last 31 d of the study. De novo lipid biosynthesis was determined by measuring the rate of incorporation of deuterium oxide into fatty acids in adipose tissue triglycerides (TG) (Jones 1996 , Leitch and Jones 1991 and 1993 ). The deuterium dose was higher than normal to ensure that adequate enrichment into adipose tissue TG was obtained. At dissection, adipose tissue not reserved for other purposes (see below) was pooled and frozen. Lipids were extracted subsequently using chloroform/methanol (2:1, v/v) and the lipid classes separated by silica gel TLC. The gel section containing TG was scraped from the TLC plate and eluted with a hexane/chloroform/diethyl ether mixture (5:2:1, v/v/v). After removal of the solvent, the samples were combusted in Pyrex tubes containing 0.5 g copper oxide and a 2.5 cm length of 1-mm diameter silver wire. The resulting water was vacuum distilled in Vycor tubes containing 75 mg of zinc reagent (Biogeochemical Isotope Laboratories, Indiana University, Bloomington IN) and reduced to hydrogen gas at 500°C for isotope enrichment measurements. Deuterium enrichment of urinary water was determined after dilution and vacuum distillation. Hydrogen isotope abundance was measured on a Finnigan MAT 252 Gas Isotope Ratio Mass Spectrometer (ThermoQuest, San Jose, CA) equipped with an automatic tube cracker, after reduction of water to hydrogen gas (Delany et al. 1989 ).

The rate of endogenous fatty acid synthesis and incorporation into triglyceride (TG) was calculated on the basis of a linear model (Hems et al. 1975 ) to calculate fractional synthetic rate (FSR):

where del_TG and del_urine reflect the per mil (o/oo) change in deuterium enrichment in TG and urine water above baseline, respectively. The fractional enrichment of urine is used as a measure of plasma enrichment. Due to the high expected deuterium enrichment, the baseline deuterium enrichments were assumed to be negligible. In addition, the ratio was adjusted to account for carbon atoms in glycerol (GLY) using a hypothetical TG with three 17-carbon monounsaturated fatty acids (FA):

where D is the number of deuterium atoms per fatty acid molecule and C and H reflect the carbon and hydrogen atoms, respectively.

The deuterium enrichment in the urine samples was very high, necessitating dilution by 1:200. There was no difference in urine deuterium enrichment between control and CLA-treated mice (276,897 ± 14,252 vs. 279,638 ± 14,252 o/oo). The deuterium enrichment of adipose tissue fatty acids was much higher than anticipated or normally measured using gas isotope ratio mass spectrometry. Therefore, we performed a linearity test, which revealed that the measurements through the range of enrichment found in these studies were highly linear (r² = 0.9999) to >30,000 o/oo, with an intercept of nearly zero (18.8 o/oo).

Dissections, blood collection and plasma analyses.

At the end of the 5-wk experimental period and after 2–3 h of food deprivation, the mice were anesthetized with isoforane and blood taken by heart puncture. The mice were then killed by cervical dislocation while still anesthetized. The carcasses were dissected and the following tissues were removed, weighed and quick frozen in liquid nitrogen for later analysis of uncoupling gene expression: left epididymal adipose depot, left kidney, interscapular brown adipose tissue (BAT) and abdominal muscle. The abdominal muscle was taken from the lower left abdominal wall and included the internal and external abdominal oblique, rectus abdominis and tranversus abdominus muscles. In addition, the remaining adipose depots, right epididymal, left and right inguinal, left and right retroperitoneal, and the mesenteric depot were removed, weighed and quick frozen for measurement of deuterium enrichment in lipid. RIA were performed on plasma to measure insulin and growth hormone levels (Rat Insulin Kit #RI-13K; Linco Research, St. Charles, MO; and rat growth hormone assay system #RPA551 from Amersham Life Science, Princeton, NJ). A Colormeteric Hexokinase Glucose assay for plasma glucose was also completed (Glucose HK Kit #16–10 from Sigma Diagnostics, St. Louis, MO).

Uncoupling protein expression analysis.

Expression of uncoupling proteins 1, 2 and 3 (UCP1, UCP2 and UCP3) was assessed in specific tissues by Northern blot. Total RNA from white adipose tissue was extracted in 4 mol/L guanidinium isothiocynate and pelleted through cesium chloride (Chirgwin et al. 1979 ). Total RNA from kidney, abdominal muscle and BAT was isolated using Trizol reagent (#15596–026, Life Technologies, Rockville, MD) as described (Chomczynski and Sacchi 1987 ). PolyA RNA was prepared from kidney, muscle and BAT total RNA using oligodT (Aviv and Leder 1972 ) bioton:streptavidin magnetic bead technology (Kit #Z5310 Promega, Madison, WI).

Uncoupling protein expression was assessed by Northern blot in abdominal muscle, kidney, interscapular BAT and epididymal white adipose tissue as follows: UCP2 and 3 expression were measured in abdominal muscle of CLA-treated (n = 5–6) and control (n = 5–6) mice; UCP 2 expression was assessed in kidney (5 treated, 4 controls); UCP1 and UCP2 expression were measured in BAT for 4 treated and 5 control mice; and UCP2 expression was measured in epididymal fat from 4 treated and 4 control mice (data not shown).

DNA, from several plasmid restriction fragments, served as probes for Northern blots. We purified a 1.2-kb insert from a rat cDNA UCP1 clone (given by Dr. D. Ricquier, Centre de Recherche sur l’Endocrinologie Moleculaire et le Development, Meudon, France), a 1.2-kB insert encoding the 5' end of mouse cDNA UCP2 (Image Consortium clone #747587), and a 700-bp insert encoding the 5' end of mouse cDNA UCP3 (given by Dr. L. Kozak, Pennington Biomedical Research Center, Baton Rouge, LA). Inserts were random primed (Feinberg and Vogelstein 1983 ) using (-³²P) dATP and random decamers (Ambion Kit #1455, Autin, TX) to a specific activity of 1–2 x 10⁹ dpm/µg DNA. For Northern blot hybridization, RNA was electrophoresed through a 1.4% agarose gel using a formaldehyde buffer and transferred to Zeta-probe GT nylon membrane (#162–0192, Biorad, Hercules, CA) in 10X SSC via upward capillary transfer (Sambrook et al. 1989 ). Blots were hybridized and washed according to specifications in the Zetaprobe manual using a hybridization buffer containing 50% formamide (Church and Gilbert, 1984 ). Hybridization signals were quantified by phosphoimage analysis using Image Quant Software version 3.3 (Molecular Dynamics, Sunnyvale, CA) and normalized to 18S and/or ß-actin. Permanent images were made by exposure to Kodak BioMax film (Eastman Kodak, Rochester, NY).

Total RNA blots prepared from interscapular BAT were hybridized with both the UCP1 and UCP2 probes. PolyA RNA Northern blots from abdominal muscle were hybridized with both the UCP2 and UCP3 probes. PolyA RNA from epididymal fat and kidney was hybridized with the UCP2 probe only. A total of 4–6 samples of each tissue from the control and CLA treatment group were analyzed.

Statistical analysis.

Statistical evaluation for differences among group means were performed by ANOVA. If a main effect was observed, post-hoc comparisons were made using a t test adjusted for multiple comparisons. For the plasma insulin and leptin assays, fatty acid biosynthesis and Northern blots, a simple Student’s t test was done to evaluate significance between control and CLA treated groups. Differences were considered significant when P < 0.05.

	RESULTS

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CLA had no effect on body weight in this experiment (Fig. 1 ). Overall energy intake was not affected by CLA treatment (Fig. 2 ).

View larger version (16K): [in this window] [in a new window]   Figure 1. Body weight of mice fed a high fat diet containing 1% conjugated linoleic acid (CLA) or no CLA. Mice were weighed twice each week and the data were averaged into a weekly mean for each group. Each point represents the mean ± SEM, n = 8.

View larger version (11K): [in this window] [in a new window]   Figure 2. Cumulative energy intake of mice fed a high-fat diet with 1% conjugated linoleic acid (CLA) or no CLA. Food intake was weighed twice each week. Each point represents the mean ± SEM, n = 8.

View larger version (15K): [in this window] [in a new window]   Figure 3. Weights of four adipose depots (inguinal, epididymal, retroperitoneal and mesenteric) in mice fed a high fat diet containing 1% conjugated linoleic acid (CLA) or no CLA. For all depots except the mesenteric depot, the data are presented as the average of the left and right depot weights. Each bar represents the mean ± SEM, n = 8. Differences from the control group are indicated by an asterisk (*=P < 0.05; **=P < 0.001).

View larger version (51K): [in this window] [in a new window]   Figure 4. Energy expenditure (24 h) of mice fed a high fat diet containing 1% conjugated linoleic acid (CLA) or no CLA measured weekly over a 6-wk period. The baseline measure was taken in both groups of mice before random assiginment into CLA-treated and control groups. For each time point, equal numbers of control and treated mice were tested simultaneously. Panel A: energy expenditure (EE) during the day ± SEM Each bar represents the mean ± SEM, n = 8. The overall ANOVA indicated a significant difference between treated and control groups (P < 0.001). Comparing groups at each week with the post-hoc analysis, the only significant difference occurred in wk 2 (P < 0.005). Panel B: energy expenditure during the night ± SEM Overall, there was a significant effect of CLA on nighttime EE (P < 0.001). A significant difference was shown for night EE in wk 4 and 5 (P < 0.05).

Representative Northern blots for the assessment of UCP gene expression are shown in Figure 5A , B , C , D . There were no significant effects of CLA treatment on UCP gene expression in skeletal muscle, epididymal adipose tissue or kidney. In BAT, UCP1 expression was not affected by CLA treatment. However, UCP2 expression in BAT, although very low, was increased by 50% by dietary CLA (P < 0.01; t test). Expression of UCP2 in BAT was highly variable as assessed using Northern blots against total RNA. Therefore, these specific results should be considered preliminary.

View larger version (42K): [in this window] [in a new window]   Figure 5. Northern blot analyses of uncoupling protein 1, 2 and 3 (UCP1, 2 and 3) gene expression in mice fed a high fat diet containing 1% conjugated linoleic acid (CLA) or no CLA. Blots for two treated mice [T] and two controls [C] are presented. Panel A: UCP1 and UCP2 expression and 18S ribosomal RNA in brown adipose tissue (BAT). Panel B: UCP2 and ß-actin expression in the kidney. Panel C: UCP3 and ß-actin expression in abdominal muscle. Panel D: UCP2 and ß-actin expression in abdominal muscle. There were no significant effects of CLA treatment except for a slight increase in UCP2 expression, normalized to 18S RNA in BAT (P < 0.05; t test).

	DISCUSSION

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CLA increased EE by an average of 7.7% over the 5-wk treatment period. The total energy intake over the study period in the control group was 2.68 x 10⁶ MJ. If 7.7% of this energy were oxidized rather than stored, then 205 kJ would have been expended. Assuming minimal energy required to store circulating lipid as TG in adipose tissue, and 37.6 kJ/g of TG, this amount of energy would be equivalent to 5 g of TG. The difference in the summed weight of the inguinal, epididymal, retroperitoneal and mesenteric adipose depots between control and CLA-treated groups was 1.7 g. Because these depots account for <50% of stored carcass lipid in the mouse, the total estimated TG store lost due to CLA treatment was at least 3.4 g. Therefore, the increased EE as measured by indirect calorimetry was sufficient to account for the decreased adipose depot lipid stores in CLA-treated mice.

The effect of CLA to reduce carcass lipid content was not due to a decreased de novo biosynthesis of fatty acids. In fact, when the data were presented per mouse, CLA-treated mice had higher de novo fatty acid biosynthesis than controls. It should be noted that, with the long period of labeling used in this study to measure de novo fatty acid biosynthesis, the transport of fatty acids synthesized by the liver and subsequently incorporated into adipose tissue could be a factor. Therefore, the enrichment measures reported here are more likely a reflection of synthesis in other tissues including liver as well as adipose tissue. The data suggest that the total-body fatty acid synthesis and resulting incorporation of these fatty acids into adipocyte triglyceride stores was not inhibited by CLA. Further studies with shorter labeling periods will be necessary to determine whether there are differential effects of CLA on fatty acid synthesis in different tissues. Sartory and Smith (1999) reported recently that CLA inhibited differentiation of 3T3L1 cells and also stimulated lipid biosynthesis from ¹⁴C-labeled glucose in differentiated 3T3L1 cells. The effect of CLA to slow differentiation in their report may have been nonspecific because linoleic acid had a similar effect. In the data presented here, the fractional synthetic rate of fatty acids was greater in CLA-treated mice, consistent with the findings of Satory and Smith (1999) . Normalizing lipid biosynthesis to the total amount of lipid indicated that the total synthetic rate was not affected by CLA treatment. However, assuming that the adipocyte number was the same in both CLA-treated and control mice, the de novo biosynthesis of fatty acid and incorporation into adipocyte lipid would account for a greater fraction of the total lipid per cell in CLA-treated mice. This metabolic effect of CLA could be compensatory in response to the loss of adipocyte lipid due to the stimulation of lipolysis or the inhibition of lipoprotein lipase activity as reported by Park et al. (1997) .

There was no effect of CLA on RQ, a measure of the proportion of fat energy vs. carbohydrate energy that is burned. We reported previously that CLA decreased the RQ (West et al. 1998 ). However, this effect of CLA on RQ was significant only in mice fed a low fat diet and was significant only during the night when more energy was utilized from ingested high carbohydrate diet than from stored adipose TG. We concluded from this previous finding that the effect of CLA was to mobilize stored TG and that this accounted for the drop in RQ during the night because more fat was being utilized during this time period in CLA-treated than in control mice. In this study, all of the mice were fed a high fat (45% energy as fat) diet. The decreased RQ due to mobilization of stored lipid in CLA-treated mice, or the impaired uptake and therefore metabolism of ingested lipid, would be expected to be less obvious given the already low RQ due to high fat feeding.

There are a number of possible mechanisms by which CLA could increase EE. This could be due to increased basal metabolic rate, increased thermic effect of ingesting a meal (i.e., the energy associated with the ingestion, digestion, absorption and storage/utilization of ingested energy) or due to increased physical activity. Measures of total EE cannot distinguish among these mechanisms. However, in mice, uncoupling proteins are important mediators of overall energy metabolism and EE. Uncoupling protein 1 (UCP1), expressed exclusively in BAT, is a very potent modifier of EE and is critical for maintaining core body temperature in small rodents. The newly described uncoupling proteins 2 and 3 (UCP2, UCP3) also may have important effects on energy metabolism (Fleury et al. 1997 , Vidal-Puig et al. 1997 ). UCP2 is expressed in many tissues, whereas UCP3 is expressed primarily in skeletal muscle (Jezek and Garlid 1998 ). It is possible that the uncoupling proteins are involved in both the thermic effect of a meal as well as the regulation of basal metabolic rate. In this study of CLA, CLA generally had no effects on expression of any of the uncoupling proteins in any tissue examined. The only exception was that CLA treatment slightly increased UCP2 expression in interscapular BAT. However, the role of UCP2 in overall BAT thermogenesis is likely to be small. UCP2 expression is much lower in BAT than that of UCP1; ß3 adrenergic stimulation of BAT does not upregulate UCP2 expression, whereas UCP1 expression is markedly enhanced (Yoshitomi et al. 1998 ). It has been proposed that UCP2 and UCP3 are more important as regulators of fatty acid utilization than as uncouplers involved in thermogenesis because changes in expression of these genes tend to passively follow the utilization of fatty acids as fuel, and this occurs even at thermoneutrality (Samec et al. 1998 ). This could account for a recent report suggesting that differential regulation of UCP2 gene expression may be an important factor in differential sensitivity to dietary obesity in mice (Surwit et al. 1998 ). The increased expression of UCP2 in the strain resistant to dietary obesity may have been due to altered flux of fatty acid and not related causally to the resistance to dietary obesity. Overall, on the basis of the results reported here, we conclude that CLA is not increasing metabolic rate by stimulating uncoupling protein gene expression.

If the uncoupling proteins (especially UCP1 expression in BAT) are not involved in mediating the effects of CLA, then there are a variety of other explanations. For example, CLA might increase sympathetic nervous system (SNS) activity to the periphery. Such an activation of SNS activity could result in increased EE due to effects on tissues other than BAT because sympathomimetics increase metabolic rate in humans, and BAT is not found in human adults (Ratheiser et al. 1998 ). Other possible direct effects of sympathetic activation that would increase metabolic rate include increasing the heart rate, stimulation of smooth muscle contraction in the vasculature and stimulation of lipolysis in adipose tissue, which would provide fatty acids to skeletal muscle. Alternatively, CLA could be acting by increasing the energy cost of assimilating lipid or carbohydrate energy during a meal. Future studies will be designed to determine whether CLA has specific effects on the thermic effect of a meal vs. general effects on resting metabolic rate. These can be distinguished by the appropriate studies using indirect calorimetry.

Another possibility is that CLA increases physical activity. The recent report by Levine et al. (1999) that variations in nonexercise thermogenesis (assumed to be physical activity) account for different rates of fat accumulation when human subjects consume more energy suggests that changes in normal levels of physical activity could have potent effects on overall energy metabolism. The hypothesis that CLA could increase physical activity can be tested and should be completed to rule out this possible mechanism.

There were no significant effects on circulating growth hormone, insulin or glucose due to CLA treatment. However, plasma insulin was nearly doubled by CLA treatment but the levels were highly variable. The fact that the mice were deprived of food for only 2–3 h before blood collection and dissection likely accounts for the large variation in insulin and glucose measures and obscures true differences in insulin levels between control and CLA-treated mice. We have observed that CLA treatment increases circulating insulin levels in several studies, including this study (DeLany et al. 1999 and unpublished work); this suggests that CLA is inducing an insulin-resistant state as discussed previously. Growth hormone levels were not significantly different in this study (P > 0.05). However, the variability in growth hormone measures precludes any conclusion regarding alterations in growth hormone production/clearance in mediating the effects of CLA.

In summary, chronic CLA treatment results in the persistent elevation of total EE in mice and significantly decreases adipose depot lipid stores. The effect on EE was apparent throughout the 5-wk treatment. The increased total EE induced by CLA is sufficient to account for the difference in lipid stores between treated and control mice. As we reported previously, CLA does not act by suppressing energy intake. The effect of CLA to reduce carcass lipid content is not attributable to decreased de novo fatty acid biosynthesis in adipose tissue. In addition, the increased total EE is not likely due to increased UCP gene expression, which might mediate changes in basal metabolic rate or the thermic effect of feeding. Therefore, although we cannot conclude which component of EE is affected by CLA treatment, we have ruled out a role for two mechanisms in the whole animal, i.e., UCP and de novo fat biosynthesis. Additional studies will be required in both the whole animal and in isolated tissue preparations to define precisely the target tissues by which CLA increases EE and to define further the mechanisms by which it acts.

	FOOTNOTES

 
¹ Supported by a grant cosponsored by the National Cattleman’s Beef Association and Kraft Foods awarded to D.B.W. and J.P.D.

³ Abbreviations used: BAT, brown adipose tissue; CLA, conjugated linoleic acids; EE, energy expenditure; FSR, fractional synthetic rate; RQ, respiratory quotient; SNS, sympathetic nervous system; TG, triglyceride; UCP, uncoupling protein.

Manuscript received November 12, 1999. Initial review completed December 9, 1999. Revision accepted May 12, 2000.

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TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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