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Changes in Body Composition with Conjugated Linoleic Acid

Pennington Biomedical Research Center, 6400 Perkins Road, Baton Rouge, Louisiana

Address reprint requests to: James P. DeLany, PhD, Pennington Biomedical Research Center, 6400 Perkins Road, Baton Rouge, LA 70808. E-mail: delanyjp{at}pbrc.edu.

	ABSTRACT

TOP ABSTRACT INTRODUCTION GENERAL PROTOCOL DIETARY FAT STUDIES DOSE RESPONSE AND TIME... METABOLIC RATE TIME COURSE... EFFECTS OF CLA IN... DOSE RESPONSE AND TIME... METABOLIC RATE TIME COURSE... CONCLUSION REFERENCES

 
Conjugated linoleic acid has been shown to reduce body fat accumulation in several animal models. We have conducted several studies in AKR/J mice showing that CLA reduces body fat accumulation whether animals are fed a high-fat or low-fat diet, with no effect on food intake. One mechanism by which CLA reduces body fat is by increased energy expenditure, which is observed within one week of CLA feeding and is sustained for at least six weeks. The increased energy expenditure is sufficient to account for the decreased fat accumulation. Increased uncoupling protein gene expression does not appear to be involved in the increased energy expenditure. We have observed increased fat oxidation but no decrease in de novo fat biosynthesis with CLA feeding. We have also observed increased liver weights and plasma insulin levels with higher doses of CLA. In all of the studies we have conducted to date we have used a CLA preparation that contains several isomers, primarily c9,t11 and t10,c12. It was assumed that the active form was c9,t11, as CLA was identified as an anticarcinogenic compound from cooked beef, of which the c9,t11 form accounts for 60% to 80% of the CLA. Most of the studies conducted so far must be repeated using the purified isomers in order to determine which isomers are responsible for each of the identified actions of CLA.

Key words: conjugated linoleic acid, body fat, energy expenditure, fat oxidation, lipogenesis

Abbreviations: CLA • conjugated linoleic acid • UCP • uncoupling protein • EE • energy expenditure • RQ • respiratory quotient • FSR • fractional synthetic rate

Key teaching points:

• Conjugated linoleic acid (a mixed isomer preparation) feeding causes a reduction in body fat accumulation.

• One mechanism by which CLA reduces body fat is by increasing energy expenditure.

• Increased fat oxidation appears to be involved in the decreased fat accumulation, but decreased de novo fat biosynthesis is not.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION GENERAL PROTOCOL DIETARY FAT STUDIES DOSE RESPONSE AND TIME... METABOLIC RATE TIME COURSE... EFFECTS OF CLA IN... DOSE RESPONSE AND TIME... METABOLIC RATE TIME COURSE... CONCLUSION REFERENCES

 
Conjugated linoleic acid (CLA) is a group of dienoic derivatives of linoleic acid produced by bacteria in the ruminant gut [1]. The major dietary sources of CLA for humans are beef and dairy products [2]. The anticarcinogenic effects of CLA have been studied for some time [3]. In addition, CLA appears to be protective against atherosclerosis in rabbits [4] and to partially overcome the catabolic responses due to endotoxin injection [5]. More recently, CLA has been shown to reduce body fat in mice [6], as well as rats and chickens [7]. Evaluation of the metabolic effects of CLA in both intact animals and in adipocyte culture has suggested that CLA directly affects key enzymes and processes involved in lipid mobilization and storage [6].

We have undertaken several studies to further characterize the metabolic effects of CLA in the mouse. We have extended the original studies in mice to show that CLA effectively reduces body fat in another mouse strain (AKR/J in addition to the ICN strain) [8]. Furthermore, we have shown that CLA reduces body fat in animals fed both a low- and a high-fat diet, the reduction varies for adipose depots from different sites, and CLA acts by increasing energy expenditure [8]. We demonstrated that CLA feeding produced rapid, sustained reductions in fat accumulation at relatively low doses without any major effects on food intake [9]. The increase in energy expenditure was observed within one week of CLA feeding and was sufficient to account for the decreased body fat stores in the CLA treated animals [10].

	GENERAL PROTOCOL

TOP ABSTRACT INTRODUCTION GENERAL PROTOCOL DIETARY FAT STUDIES DOSE RESPONSE AND TIME... METABOLIC RATE TIME COURSE... EFFECTS OF CLA IN... DOSE RESPONSE AND TIME... METABOLIC RATE TIME COURSE... CONCLUSION REFERENCES

 
Male, inbred AKR/J mice were obtained from the Jackson Laboratory (Bar Harbor, ME) at five weeks of age and habituated to individual housing in hanging stainless-steel cages in a room maintained at an ambient temperature of 25±1 degree C. Initially the mice were maintained on ad libitum Purina Rodent Chow (Diet #5001; Ralston Purina) and water. Mice were then switched to defined formula diets, with or without added CLA. The CLA was obtained from Nu Chek Prep, Inc. (Elysian, MN) with a reported composition of 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 14.1% remainder. The defined pelleted diets were placed onto the bottom of the cages and replaced with fresh diet when the mice were weighed and food intake (corrected for spillage and measured to 0.1g) measured three times each week.

At the end of each study, after a three-hour fast, the mice were killed by cervical dislocation and decapitated. Truncal blood was collected and plasma frozen for later measurement of insulin and leptin by radioimmunoassay (Linco Research, Inc., St. Louis, MO). Plasma glucose was measured in the control and 1.0% CLA groups by a colorimetric hexokinase glucose assay (Sigma Diagnostics, St. Louis, MO). Selected adipose depots and organs were removed and weighed. The liver and spleen of control and 1% CLA groups were sectioned and fixed in 10% formalin or glutaraldehyde for histopathological examination by light microscopy and transmission electron microscopy. Histopathological analysis was performed by two separate laboratories: the Pathology Department at the LSU Veterinary School of Medicine (Baton Rouge, LA) and the Experimental Pathology Laboratories, Inc. (Herndon, VA).

	DIETARY FAT STUDIES

TOP ABSTRACT INTRODUCTION GENERAL PROTOCOL DIETARY FAT STUDIES DOSE RESPONSE AND TIME... METABOLIC RATE TIME COURSE... EFFECTS OF CLA IN... DOSE RESPONSE AND TIME... METABOLIC RATE TIME COURSE... CONCLUSION REFERENCES

 
At six weeks of age, the mice were placed on a high-fat (45% kcal fat) or low-fat (15% kcal fat) diet with or without CLA (2.46mg CLA/kcal of diet; 1% by weight for the low-fat diet and 1.2% by weight in the high-fat diet) for six weeks. The CLA dosage was based upon previous reports in the mouse using CLA [6],7]. Sample size was 8 to 10 mice/diet and treatment group. During the sixth week following the start of the defined diets, each animal was placed into a metabolic chamber for the measurement of 24hr carbon dioxide production and oxygen consumption by indirect calorimetry. The system used for the assessment of metabolic rate in the mouse has been previously described [11]. After six weeks of the diet and CLA manipulations, blood and tissues samples were obtained for analyses as described above.

The data are presented in the figures and text as mean± standard error of the mean. Body weight, energy intake, organ and adipose depot weights and carcass composition data were analyzed by analysis of variance and post hoc comparisons were made using a protected least significant difference test using SAS release 6.12. Energy expenditure observations were modeled as repeated measures on each subject with diet, CLA, chamber number, and day/night as explanatory variables arranged as a 2x2x4x2 factorial treatment structure (the highest order interaction term was not included). Body weight was included as a covariate in the model for energy expenditure. A similar model was adopted for observations of respiratory quotient, but body weight was not included as a covariate in that model. Solutions were obtained using restricted maximum likelihood estimation as implemented in MIXED procedure of SAS release 6.12. Observed significance levels for multiple comparisons of estimated treatment means (least squares means) were adjusted using the Tukey joint estimation procedure.

	DOSE RESPONSE AND TIME COURSE STUDIES

TOP ABSTRACT INTRODUCTION GENERAL PROTOCOL DIETARY FAT STUDIES DOSE RESPONSE AND TIME... METABOLIC RATE TIME COURSE... EFFECTS OF CLA IN... DOSE RESPONSE AND TIME... METABOLIC RATE TIME COURSE... CONCLUSION REFERENCES

 
Individually housed male AKR/J mice were switched to a high-fat diet (45% kcal fat) 10 days prior to being randomly assigned to treatment diets. This period of adaptation to the high-fat diet was used in both studies described here to minimize the effect of high-fat diet-induced hyperphagia in the treatment groups at study initiation [8].

In a dose response study, 60, six-week-old mice were randomly assigned to five treatment groups (n=12/group) for feeding varied doses of CLA (0%, control; 0.25%; 0.50%; 0.75%; and 1.0% CLA by weight) in a high-fat diet (45 kcal % fat). Food intake and body weight were measured three times per week from seven days prior to the start of experimental diet feeding and continued throughout the 39 days of CLA feeding.

In a time-course study, 40 mice were used as controls and fed an unadulterated high-fat diet, while 40 treatment animals were fed the high-fat diet containing 1% CLA. Measurements of food intake and body weight were made twice per week using the same protocol as described above.

Eight animals from the control group and eight from the treatment group were killed after 2, 4, 6, 8, and 12 weeks of CLA treatment. At the end of the study, blood and tissue samples were collected as described above. Livers and spleens were fixed for histopathological examination and carcasses analyzed for composition at the 12 week time point only. Plasma insulin and leptin were measured at all time points, while glucose was measured in the control and treatment group for the 12-week time point only.

Statistical evaluation for differences among group means was performed by an analysis of variance. If a main effect was observed, post hoc comparisons using a Tukey-Kramer adjustment were made. A Wilcoxon rank sum test was used to determine differences in liver lipid accumulation. For the time-course study, repeated measures were performed with respect to time using a covariance structure. All data are presented as mean±SE.

	METABOLIC RATE TIME COURSE AND MECHANISM STUDIES

TOP ABSTRACT INTRODUCTION GENERAL PROTOCOL DIETARY FAT STUDIES DOSE RESPONSE AND TIME... METABOLIC RATE TIME COURSE... EFFECTS OF CLA IN... DOSE RESPONSE AND TIME... METABOLIC RATE TIME COURSE... CONCLUSION REFERENCES

 
Four-week-old AKR/J male mice were initially fed ad libitum Purina Mouse Chow (Diet #5015, Ralston Purina). Prior to treatment with dietary CLA, mice were switched to a high-fat diet (45% kcal fat) for an adaptation period of 10 days. Mice were then maintained on the high fat diet (n=8) or switched to a high fat diet containing 1% CLA (n=8). Mice were provided ad libitum access to pre-weighed defined diets in pellet form placed on the bottom of each cage. Twice per week the animals and remaining diet were weighed and fresh pre-weighed diet was given. Food intake was corrected for spillage and all measurements were to the nearest 0.1g.

The system used for the assessment of metabolic rate in the mouse has been previously described [8]. During the 10 days of high-fat diet adaptation, the 16 male six-week-old AKR/J mice were also habituated to the metabolic chamber conditions by placing each animal in the metabolic cage for one 24-hour period without collecting data. At the end of the dietary habituation period, mice were placed into the metabolic chambers for the measurement of baseline energy expenditure prior to CLA treatment. Metabolic rate measurements were taken on each mouse once per week for five weeks.

Mice were given drinking water containing deuterium oxide for the last three weeks 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 [12,13]. At dissection, adipose tissue not reserved for other purposes was pooled and frozen. The enrichment of deuterium in isolated triglyceride fatty acids was measured for deuterium enrichment to calculate the rate of endogenous triglyceride synthesis.

Expression of uncoupling proteins 1, 2 and 3 (UCP1, UCP2 and UCP3) was assessed in specific tissues by Northern blot. For Northern blot hybridization, RNA was electrophoresed through a 1.4% MOPS formaldehyde gel and transferred to Zeta-probe GT nylon membrane (#162-0192, Biorad) in 10X SSC via upward capillary transfer. Blots were hybridized and washed according to specifications in the Zetaprobe manual using a hybridization buffer containing 50% formamide [14]. Hybridization signals were quantified by phosphorimage analysis using Image Quant Software version 3.3 (Molecular Dynamics) and normalized to 18S and/or ß-actin.

Statistical evaluations for differences among group means were performed by an analysis of variance. If a main effect was observed, post hoc comparisons were made. For the plasma insulin and leptin assays, fatty acid biosynthesis, and Northern blots, a simple Student t test was done to evaluate significance between control and CLA treated groups.

	EFFECTS OF CLA IN MICE FED HIGH- AND LOW-FAT DIETS

TOP ABSTRACT INTRODUCTION GENERAL PROTOCOL DIETARY FAT STUDIES DOSE RESPONSE AND TIME... METABOLIC RATE TIME COURSE... EFFECTS OF CLA IN... DOSE RESPONSE AND TIME... METABOLIC RATE TIME COURSE... CONCLUSION REFERENCES

 
Body weights were lower in CLA treated mice fed either a high-fat or low-fat diet (Fig. 1). CLA treatment significantly reduced each adipose depot weight relative to control diets for both the high-fat and low-fat diet groups (Fig. 2, p<0.001 for all comparisons). CLA treatment reduced adipose depot weights by approximately the same percentage relative to same-diet controls in both the high-fat and low-fat diet groups. CLA treatment had the largest effect on the retroperitoneal adipose depot; reducing it by 78.2% and 87.7% in the high-fat and low-fat CLA treatment groups respectively. Increased liver and the spleen weights were observed with CLA treatment, independent of diet group and body weight (p<0.0001 and p<0.014 for liver and spleen respectively).

View larger version (38K): [in this window] [in a new window]   Fig. 1. Body weights for control and CLA treated mice fed high-fat or low-fat diets for 6 weeks. CLA significantly reduced body weight gain in the high-fat (*) and low-fat () diet groups (p<0.001).

View larger version (45K): [in this window] [in a new window]   Fig. 2. Adipose depot weights for mice control and CLA treated mice fed high- or low-fat diets. CLA treatment significantly reduced adipose depot weights in the high-fat (*) and low-fat () diet groups (p<0.001).

Energy expenditure (EE) data were adjusted for body weight differences using body weight as a covariate (Fig. 3). There was a statistically significant increase in the total EE in CLA treated animals when both diet groups were combined (p<0.0067), or when examining either diet individually (p<0.05). This increased EE occurred despite a reduced total body mass and a marginally reduced energy intake in these animals compared with controls.

View larger version (56K): [in this window] [in a new window]   Fig. 3. Energy expenditure after 6 weeks of CLA treatment for mice fed a high-fat or a low-fat diet. CLA treatment significantly increased energy expenditure in the high-fat (*) and low-fat () diet groups (p<0.01).

View larger version (28K): [in this window] [in a new window]   Fig. 4. Respiratory quotient after 6 weeks of CLA treatment in mice fed a high-fat or low-fat diet. CLA treatment blocked the increased RQ observed during the feeding period (night), indicating increased fat oxidation.

	DOSE RESPONSE AND TIME COURSE STUDIES

TOP ABSTRACT INTRODUCTION GENERAL PROTOCOL DIETARY FAT STUDIES DOSE RESPONSE AND TIME... METABOLIC RATE TIME COURSE... EFFECTS OF CLA IN... DOSE RESPONSE AND TIME... METABOLIC RATE TIME COURSE... CONCLUSION REFERENCES

At the end of treatment, there was a dose dependent effect of CLA on all adipose depots relative to the controls (Fig. 5). The adipose depot most sensitive to the effects of CLA was the retroperitoneal, with a significant reduction in depot weight observed at 0.50%, 0.75% and 1.0% CLA (p<0.01 for all comparisons). The inguinal, epididymal and mesenteric adipose depots showed a significant difference at the 0.75% and 1.0% doses (p<0.05). Body composition data supported the adipose depot weight data for the effect of CLA to reduce body fat. Body lipid content, in absolute terms and as a percentage, was significantly reduced at the 0.50%, 0.75% (p<0.05) and 1.0% CLA dose (p<0.01). There was a trend for increased % body protein content with higher doses of CLA, which was significant at the 1.0% dose (p<0.01) compared to controls. Carcass ash content was not affected by CLA treatment.

View larger version (36K): [in this window] [in a new window]   Fig. 5. Adipose depot weights in mice fed increasing doses of CLA in a high-fat diet. *Cumulative adipose depot weight was reduced with 0.5% CLA (p<0.01). The retroperitoneal depot was the most responsive to CLA treatment.

Plasma leptin levels tended to decrease with the higher CLA doses, but there were no significant differences. In contrast, plasma insulin showed increased levels with higher doses of CLA, becoming significant from control in the 1.0% group (p<0.05). Plasma glucose levels were not significantly affected by CLA treatment (145±9 for controls and 153±11 mg/dL for 1% CLA).

Time-Course Study
Body weight was significantly reduced by day 22 of 1% CLA feeding, when compared to controls, and remained lower throughout the 12 weeks of treatment (p<0.005). However, as the study progressed, the difference between control and CLA treatment was diminished, particularly by 12 weeks (Fig. 6). Cumulative energy intake was not affected by 12 weeks of 1% CLA treatment. As found in the dose response study, the adipose depot most sensitive to CLA treatment was the retroperitoneal, with significant reductions in weight at all time points (p<0.005, Fig. 7). The inguinal and epididymal fat depots were significantly lower than control at week 4 and through week 12 of the study (Fig. 7). Mesenteric adipose depot weights were reduced by CLA treatment, but the effects of CLA on this depot were not apparent until week 6.

View larger version (13K): [in this window] [in a new window]   Fig. 6. Body weights of control and 1% CLA treatment over 12 weeks. *CLA treatment significantly reduced body weight by 4 weeks of treatment (p<0.001)..

View larger version (40K): [in this window] [in a new window]   Fig. 7. Adipose depot weights for control and CLA treated mice fed a high-fat diet. *Cumulative adipose depot weight was reduced after 4 weeks of CLA treatment (p<0.001). The retroperitoneal depot was reduced after 2 weeks (p<0.001), the inguinal and epididymal after 4 weeks (p<0.05), and the mesenteric after 6 weeks (p<0.001) of CLA treatment.

As we have observed in previous studies, liver weights were significantly higher in the 1% CLA group compared to controls, beginning at week 2 and continuing through week 12. CLA treatment also increased the weight of the spleen, with a significant difference observed at the 8-week time point (p<0.01), but not at the 12-week time point (p<0.09). Histopathological examination of livers and spleens from control and 1% CLA treated animals at the 12-time point confirmed the results in the dose ranging study. Mild (grade=2) to moderate (grade=3) centrilobular fatty changes were observed, which was higher (p<0.05) in livers from the CLA group (2.75±0.27) compared to controls (1.67±0.31). Lymphoid hyperplasia was observed in spleens, which was similar in the control and CLA groups.

Plasma insulin levels showed a tendency to increase in the CLA treated animals over time, being approximately 2 times that in controls at the 8-week time point (p<0.05) and even higher at the 12-week time point (p<0.001). Plasma leptin levels in the CLA treated were significantly reduced at the 6-week time point (p<0.001), but were not different from control values at the eight- and 12-week time points. Plasma glucose levels were not significantly different between CLA treated and control animals at the 12-week time point (207±13 in controls vs. 223±9 in CLA group).

	METABOLIC RATE TIME COURSE AND MECHANISM STUDIES

TOP ABSTRACT INTRODUCTION GENERAL PROTOCOL DIETARY FAT STUDIES DOSE RESPONSE AND TIME... METABOLIC RATE TIME COURSE... EFFECTS OF CLA IN... DOSE RESPONSE AND TIME... METABOLIC RATE TIME COURSE... CONCLUSION REFERENCES

 
Overall energy intake was not affected by CLA treatment. There was a trend for CLA treatment to reduce body weight over time as compared to controls; however, this was not statistically significant. Although there was no significant effect on body weight or energy intake by CLA treatment, CLA treatment reduced adipose depot weights, which was significant in the inguinal (p<0.05), epididymal (p<0.05) and the retroperitoneal (p<0.001), but not in the mesenteric depot.

As seen in our first study, there was a significant overall effect of CLA on 24-hour EE (p=0.012, Fig. 8). The average increase in total EE due to CLA was approximately 7.7% above the control group. The increase in EE was observed during the first week of CLA treatment for both daytime and nighttime measures, although there was inter-week variation in the magnitude of the CLA effect. During the daytime, the most significant effect of CLA treatment was observed during week 2, while for nighttime EE the most significant effects were seen during weeks 4 and 5 of treatment.

View larger version (18K): [in this window] [in a new window]   Fig. 8. Energy expenditure of control and 1% CLA treated mice over 5 weeks. Overall, CLA treatment significantly increased daytime and nighttime energy expenditure (p<0.05).

There were no significant effects of CLA treatment on uncoupling protein gene expression in skeletal muscle, epididymal adipose tissue or kidney. In brown adipose tissue, UCP1 expression was not affected by CLA treatment. However, UCP2 expression in brown adipose tissue, although very low, was increased by approximately 50% (p<0.01).

	CONCLUSION

TOP ABSTRACT INTRODUCTION GENERAL PROTOCOL DIETARY FAT STUDIES DOSE RESPONSE AND TIME... METABOLIC RATE TIME COURSE... EFFECTS OF CLA IN... DOSE RESPONSE AND TIME... METABOLIC RATE TIME COURSE... CONCLUSION REFERENCES

 
Our observations that CLA fed as a dietary admixture reduces body fat content in mice is consistent with other published studies [6,7]. We have demonstrated that CLA reduces body fat content in the AKR/J mouse strain as well as in the ICN strain. It is clear that the reduced fat accumulation associated with CLA treatment is not due to decreased energy intake. In addition, we have shown that these effects are independent of dietary fat content, as CLA reduced body fat content to approximately the same extent in animals fed a high-fat or a low-fat diet. The effects of CLA on reduced fat accumulation are not universal, as the retroperitoneal adipose depot weight was reduced the most by CLA treatment while the epididymal depot was reduced in size to a lesser extent. The specific mechanisms for the differential regional response to CLA treatment are unknown.

We have now shown in two studies that CLA treatment increases energy expenditure despite causing a reduction of body lipid stores. This is in contrast to the reduced metabolic rate normally observed with a loss of body weight due to reduced energy intake [15]. We have demonstrated that the increased energy expenditure with CLA treatment is sufficient to account for the decreased fat accumulation [10]. The mechanisms by which CLA could promote an increase of energy expenditure are numerous. It could be acting through stimulation of the autonomic nervous system, through direct effects on metabolism in specific tissues or organs or through effects on energy substrate flux and availability. We have demonstrated that CLA has little effect on UCPs, so that this is probably not a mechanism by which CLA increases energy expenditure. However, we have not measured UCP protein levels or purine nucleotide binding.

Little work has been done on the mechanisms by which CLA might affect energy metabolism. However, a recent report using isolated adipocytes suggests that CLA directly stimulates lipolysis and decreases lipoprotein lipase [14]. This could result in increased energy expenditure due to the increased energy needs for recycling between fatty acids and triglycerides or could shunt fatty acids into beta oxidation if storage was impaired. An increased activity of carnitine palmityl transferase in muscle of animals treated with CLA supports the latter explanation [6]. Our finding that CLA blocks the normal diurnal differences in respiratory quotient is consistent with CLA stimulating lipolysis during the night, which would provide more fatty acids for oxidation, thereby lowering RQ. We have further shown that CLA does not cause an increase in lipid synthesis, at least in animals fed a high-fat diet. However, CLA feeding increases the proportion of lipid that is stored from synthesized fatty acids. This finding further supports the findings that CLA causes a reduction of fat accumulation by increasing oxidation of dietary lipids.

Two potential negative effects of CLA feeding have been observed consistently in our studies. One is increased liver and spleen weights. The increased liver weight associated with CLA treatment is likely due to liver lipid accumulation since this has been found in a variety of dietary manipulations including rapid weight loss [16]. Similarly, since CLA has been reported to modulate immune function [17], perhaps through cytokines, it is also not surprising that spleen weight was affected. Histopathological examination of liver and spleens from control and 1% CLA treated animals have shown slightly higher lipid accumulation in the CLA group with no signs of pathology. The other negative effect of CLA feeding, particularly at high levels of CLA, is increased plasma insulin levels. This indicates that feeding high levels of CLA are inducing insulin resistance, possibly due to increased levels of free fatty acids [18].

It should be noted that the CLA used in these experiments was not pure and included a number of isomers in addition to the putative active form of the molecule (cis-9, trans-11 octadecadienoic acid). It is certainly possible that the different isomers have different metabolic effects. Further studies using pure reagents are clearly needed in order to fully understand the metabolic specificity of these different isomers of linoleic acid. In fact, a recent report has shown that the t10,c12 isomer of CLA is the effective isomer for the reduced body fat which has been observed [19]. It had been thought that the c9,t11 isomer was the effective form, since the anticarcinogen extracted from cooked beef was determined to be CLA, and approximate 60% to 80% of the naturally occurring CLA in beef is the c9,t11 isomer [20].

In conclusion, our studies have confirmed and expanded on previously reported studies of the effects of CLA on body fat accumulation in animals. We have shown that conjugated linoleic acid at a concentration of 0.5% to 1.0% (w/w) in both low-fat and high-fat diets has profound metabolic effects in mice resulting in an increase energy expenditure, a shift in the fuel mix burned, and a decrease of body fat content. The increased energy expenditure is adequate to account for the decreased fat deposition. The mechanisms for the increased energy expenditure are unknown, but increased UCP gene expression does not appear to be involved. The increased fat oxidation, observed both in vivo and in vitro can also help to explain the decreased fat accumulation, while decreased de novo fat biosynthesis does not appear to be involved.

Received February 1, 2000.

	REFERENCES

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