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Nutrient-Gene Interactions

Increasing the Amount of Fat in a Conjugated Linoleic Acid–Supplemented Diet Reduces Lipodystrophy in Mice

Division of Clinical Nutrition, National Institute of Health and Nutrition, Shinjuku-ku, Tokyo 162-8636, Japan and ^* Department of Health and Nutrition, Bunkyo University Women’s College, Chigasaki, Kanagawa 253-8550, Japan

²To whom correspondence should be addressed. E-mail: ezaki{at}nih.go.jp.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Conjugated linoleic acid (CLA) is a naturally occurring group of dienoic derivatives of linoleic acid found in beef and dairy products. However, when 1 g CLA/100 g diet was given to mice in a low fat diet (4 g fat/100 g diet), they showed a marked decrease in fat mass, but demonstrated symptoms of lipoatrophic diabetes, i.e., marked hepatomegaly and insulin resistance. In this study, to determine whether the decrease in adipose tissue was responsible for these adverse effects, mice were fed different doses of CLA and dietary fat. In Experiment 1, mice were fed different doses of CLA (0, 0.1 and 1 g CLA/100 g diet) in a fixed 4 g fat/100 g diet; in those fed 0.1 g CLA, subcutaneous white adipose tissue (WAT) weight was 48% lower than in mice fed 0 g CLA. The mice fed 0.1 g CLA did not exhibit hepatomegaly and insulin resistance. In Experiment 2, mice were fed for 5 mo different amounts of dietary fat (4 , 13 and 34 g fat/100 g diet) in 0 or 1 g CLA/100 g diet; in mice fed 1 g CLA with 34 g fat, retroperitoneal and subcutaneous WAT weights were 76 and 79% lower, respectively, than those of mice fed 0 g CLA with 34 g fat. Mice fed 1 g CLA in the diet with 34 g fat had normal plasma insulin concentrations and a 45% greater liver weight. These data suggested that the percentage of CLA in dietary fat might be a determinant of CLA-mediated lipodystrophy.

KEY WORDS: • obesity • leptin • glucose transporter 4 • peroxisome proliferator-activated receptor • sterol regulatory element-binding protein • mice

Conjugated linoleic acid (CLA) is a group of positional and geometric isomers of conjugated dienoic derivatives of linoleic acid. The major dietary sources of CLA for humans are beef and dairy products (1). CLA has received considerable attention because of its anticarcinogenic and antiatherogenic properties and its ability to reduce body fat while enhancing lean body mass (2). Reduction of body fat by CLA was observed in pigs (3), mice (4,5) and hamsters (6).

However, when 1 g CLA/100 g diet, a mixture of 33.0% cis-9, trans-11 and 34.8% trans-10, cis-12, was given to mice in a low fat diet, a marked reduction of fat mass by CLA resulted in insulin resistance and hepatomegaly (7). These metabolic abnormalities are characteristics of lipodystrophy (8,9). The decrease in fat tissue was due to apoptosis. Tumor necrosis factor- (TNF-) and uncoupling protein 2 (UCP2) mRNAs levels were 11- and 5-fold greater, respectively, in isolated adipocytes from mice fed CLA than from those not fed CLA. Because TNF- induces apoptosis of adipocytes and up-regulates UCP2 mRNA in mice, a marked increase of TNF- mRNA with an increase of UCP2 might cause CLA-induced apoptosis (7). Because continuous leptin infusion reverses hyperinsulinemia and partly prevents lipid accumulation in liver tissues, hepatomegaly was likely due in part to hypoleptinemia and hyperinsulinemia (7). We hypothesized that the marked reduction of fat mass caused hyperinsulinemia and hepatomegaly.

In human studies, 3 g/d of CLA supplementation for 2 mo did not affect body composition and energy expenditure in normal weight women (10); 3.4 g/d CLA supplementation for 3 mo reduced body fat mass in overweight and obese subjects (11); and 4.2 g/d CLA supplementation reduced body fat in men (12). These three trials did not have any overt adverse effects. These data are in agreement with our hypothesis; when we assumed that a marked decrease of fat mass contributed to CLA-induced hyperinsulinemia and hepatomegaly, the slight decrease or no change in body fat observed in human study might not have adverse effects. In contrast, in a recent randomized, double-blind controlled trial, supplementation with 3.4 g of purified trans-10, cis-12 CLA for 3 mo increased insulin resistance and glycemia with a slight reduction of fat mass in abdominally obese men. When a mixture of CLA (trans-10, cis-12 CLA and cis-9, trans-11 CLA) was given, however, no adverse effects were observed (13). In contrast to our hypothesis, these data indicated that trans-10, cis-12 CLA per se causes insulin resistance in the absence of a marked fat mass decrease.

To examine in greater detail the contribution of fat mass decrease by CLA supplementation to hyperinsulinemia and hepatomegaly, we examined the effects of CLA under two dietary conditions, i.e., a decreased amount of CLA and an increased amount of dietary fat. If we assume that a marked decrease in fat mass was a major cause of hypoleptinemia, a reduction in the amount of CLA might simply decrease fat mass moderately but not cause the adverse effects. At present, several dose-dependent studies of CLA in mice have been conducted, and a minimal effective dose of CLA to reduce body fat can be estimated. Belury et al. (5) showed that mice fed 0.5 g CLA/100 g diet in a low fat diet for 6 wk had reduced body weight but increased liver triglyceride concentration. DeLany et al. (14) reported that male AKR/J mice fed a high fat diet containing 0.5 g CLA/100 g diet for 12 wk exhibited reduced body fat but increased plasma insulin concentration. Ohnuki et al. (15) showed that Std ddY male mice fed a low fat diet containing 0.25 g CLA/100 g diet for 4 wk exhibited reduced body fat without liver triglyceride accumulation. Thus, according to these mice studies, 0.25 g CLA/100 g diet is a minimal dose with which to observe antiobesity effects. However, in these studies, insulin resistance was not evaluated by an insulin tolerance test or glucose clamp studies. Another way in which to increase adipose tissue mass is to increase dietary fat intake. We increased dietary fat while the dose of CLA was kept stable. Through these two dietary interventions, we estimated the role of a decrease in adipose tissue on CLA-induced hyperinsulinemia and hepatomegaly.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Mice.

Female C57BL/6J mice were obtained from Tokyo Laboratory Animals Science (Tokyo, Japan) at 7 wk of age and fed a normal laboratory diet (CE2, Clea, Tokyo, Japan) for 1 wk to stabilize all metabolic conditions. Each cage contained 5–6 mice. Mice were exposed to 12-h light:dark cycle and maintained at a constant temperature of 22°C. All procedures were in accordance with the National Institute of Health and Nutrition Guidelines for the Care and Use of Laboratory Animals in Japan.

Diets.

The dietary compositions used in our experiments are shown in Table 1. Safflower oil was the source of dietary fat. Safflower oil (high oleic type) contained 46% oleic acid [18:1(n-9)] and 45% linoleic acid [18:2(n-6)] of total fatty acids. CLA was kindly provided and prepared as a free fatty acid by Rinoru Oil Mills (Tokyo, Japan) and stored frozen in plastic bottles blanketed with nitrogen. Linoleic acid was isomerized to CLA with isomers (33.0% c9, t11; 34.8% t10, c12; 2.4% c9, c11/c10, c12; 2% t9, t11/t10, t12 of total fatty acids). The ingredients for the purified diets were mixed, formed into a dough with the addition of water, rolled into pellets, wrapped with Saran wrap (Asahikasei, Tokyo, Japan) and stored at -20°C until use to minimize fatty acid oxidation.

Experiment 1: dose effects of CLA.

Mice were fed different doses of CLA (0, 0.1 and 1 g CLA/100 g diet) in a fixed 4 g fat/100 g diet for 5 mo. The composition of each diet is shown in Table 1. The experiment was conducted twice. For the insulin tolerance test, 9 wk after CLA supplementation, human insulin (Humulin R, Eli Lilly Japan K.K., Kobe, Japan) was injected intraperitoneally (4.5 pmol/g body) into fed mice. Blood glucose was measured in samples obtained from the tail tip before and 15, 30, 60, 90 and 120 min after insulin injection, using a TIDEX glucose analyzer (Sankyo, Tokyo, Japan). Energy intake was measured during the wk 13–14 of feeding period, and body fat amount was estimated by dual energy X-ray absorptiometry (Lunar PIXI mus2 densitometer, Lunar Corporation, Madison, WI) during wk 19 of feeding. At the end of the experiments, fed mice were anesthetized at 1000 h by intraperitoneal injection of pentobarbital sodium (0.08 mg/g body, Nembutal, Abbot, North Chicago, IL).

Experiment 2: effects of dietary fat amount on CLA-induced lipodystrophy.

Mice were fed different amounts of dietary fat (4, 13 and 34 g fat/100 g diet) in 0 or 1 g CLA/100 g diet for 5 mo. The composition of each diet is shown in Table 1. The experiment was conducted twice. Energy intake was measured during 13–14 wk of feeding. Blood samples were obtained by cutting the tail end. In food-deprived mice, immunoreactive insulin was measured at 15 wk by an Insulin Assay Kit (Morinaga, Kanagawa, Japan). Plasma leptin was measured in fed mice at 19 wk by a Mouse Leptin Assay Kit (Morinaga), 3,5,3'-triiodothyronine (T3) by T-3 RIA Bead (Dainabot, Tokyo, Japan) and 3,5,3',5'-tetraiodothyronine (T4) by T-4 RIA Bead (Dainabot). At the end of the experiments, fed mice were anesthetized at 1000 h by intraperitoneal injection of pentobarbital sodium (0.08 mg/g body, Nembutal, Abbot). Liver, gastrocnemius, and parametrial white adipose tissue (WAT) were isolated immediately, weighed and homogenized in guanidine-thiocyanate; RNA was prepared by the method of Chirgwin et al. (17). Gene expression analysis was performed in mice fed 4 and 34 g fat/100 g diet.

Preparation of cDNA probe and Northern blot.

To study the mechanism(s) of CLA-induced hepatomegaly, we examined expression levels of several target genes of sterol regulatory element-binding protein-1 (SREBP-1), SREBP-2 and peroxisome proliferator-activator receptor (PPAR). The target genes of SREBP-1c involved in fatty acid and triglyceride synthesis include acetyl-CoA carboxylase (ACC), stearoyl-CoA desaturase-1 (SCD-1), and fatty acid synthase (FAS), whereas genes involved in cholesterol metabolism that are regulated by SREBP-2 include 3-hydroxy-3-methyglutaryl-CoA (HMG-CoA) synthase, HMG-CoA reductase, and LDL receptor (18). PPAR is a nuclear receptor that regulates the expression of enzymes involved in fatty acid oxidation such as lipoprotein lipase (LPL), acyl-CoA synthetase (ACS), and UCP2 (19,20).

cDNA clones containing the coding sequence of human UCP2 were obtained by polymerase chain reaction (PCR) amplification as described previously (21). The cDNA fragments for mouse SREBP-1, SREBP-2, HMG-CoA synthase, HMG-CoA reductase, and LPL were obtained by PCR from first strand cDNA using mouse liver total RNA as described previously (22). The cDNA probes for rat LDL receptor and ACS were kindly provided by Dr. T. Yamamoto at Tohoku University, rat ACC and FAS by Dr. N. Iritani at Tezukayama Gakuin College, PPAR by Dr. T. Osumi at Himeji Institute of Technology, and mouse SCD-1 and glucose transporter 4 (GLUT4) by Dr. Daniel M. Lane at Johns Hopkins University. These cDNAs were used as probes for Northern blots. A portion of RNA (15 µg per lane) was denatured with glyoxal and dimethyl sulfoxide and analyzed by electrophoresis in 1 g/100 g agarose gels. After transfer to nylon membranes (NEN, Boston, MA) and UV crosslinking, RNA blots were stained with methylene blue to locate 28S and 18S rRNAs and to ascertain the amount of loaded RNAs (23). The blots were hybridized overnight at 42°C with cDNAs, which had been labeled with ³²P-dCTP (NEN) by a Megaprime DNA labeling kit (Amersham Pharmacia Biotech, Buckinghamshire, England). The filters were washed several times with 1 X SSC, 0.1 g/100 g of SDS at room temperature, washed twice at 50°C and then exposed to X-ray film at -80°C. The amounts of each mRNA were quantitated with an image analyzer (BAS 2000, Fuji Film, Tokyo, Japan).

Statistical methods.

Data were analyzed by one-way (Experiment 1) or two-way (Experiment 2) ANOVA, and significant differences among means were identified by Fisher’s protected least significant difference (PLSD) test (Super ANOVA, Abacus Concepts, Berkeley, CA). When the variances were not homogeneous according to Bartlett’s test (Statview 5.0, Abacus Concepts), data were transformed logarithmically, and then the transformed data were analyzed by ANOVA followed by multiple comparison. When the variances were not homogenous even after logarithmic transformation, the results were presented as medians with range and then analyzed by Kruskal-Wallis ANOVA followed by the Kolmogorov-Smirnov two-sample test (Statview 5.0). The insulin-tolerance curve of each group in Figure 1 was compared by repeated-measures ANOVA, followed by Fisher’s PLSD test (Statview 5.0). Differences were considered significant at P < 0.05. Values in the text are means ± SEM.

View larger version (27K): [in this window] [in a new window]   FIGURE 1 Insulin tolerance tests of mice fed diets containing 4 g fat/100 g diet with 0, 0.1 or 1 g conjugated linoleic acid (CLA)/100 g diet for 9 wk (Experiment 1). Each data point represents mean ± SEM, n = 5–6 mice. Means at a time without a common letter differ, P < 0.05.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Energy intake and body weight did not differ among mice fed 0, 0.1 and 1 g CLA/100 g in a fixed amount of fat (4 g/100 g diet) (data not shown). As observed in our previous study (7), mice fed 1 g CLA, retroperitoneal and subcutaneous WAT and brown adipose tissue (BAT) were ablated, parametrial WAT weight was 73% lower, liver and spleen weights were 178 and 111% greater, respectively, than in mice fed 0 g CLA (Table 2). In mice fed 0.1 g CLA, subcutaneous WAT weight was 48% lower than in mice fed 0 g CLA. Mice fed 0.1 g CLA did not exhibit hepatomegaly and insulin resistance. The decrease in whole-body fat mass was also assessed by dual energy X-ray absorptiometry (Table 2). However, total fat in mice fed 0.1 g CLA did not differ from that in mice fed 0 g CLA, whereas total fat in mice fed 1 g CLA was 53% lower than that in mice fed 0 g CLA. Importantly, mice fed 0.1 g CLA did not exhibit hepatomegaly, whereas mice fed 1 g CLA did. Furthermore, the insulin-mediated glucose lowering effect was not impaired in mice fed 0.1 g CLA, whereas this effect was markedly impaired in mice fed 1 g CLA (P < 0.0001) (Fig. 1). Thus, a lower dose of CLA can effectively reduce body fat mass without the adverse effects after 5 mo of feeding.

In agreement with our previous observations (24), when dietary fat was increased from 4 to 34 g fat/100 g diet without CLA supplementation, mice exhibited increased parametrial, retroperitoneal and subcutaneous WAT weights in a dose-dependent manner (Table 3). As observed in our previous study with 4 g fat/100 g diet (7), mice fed 1 g CLA exhibited ablated retroperitoneal and subcutaneous WAT, decreased parametrial WAT and increased liver and spleen weights. However, mice fed increased dietary fat with or without CLA supplementation exhibited increased WAT weight. In mice fed 1 g CLA with the highest amount of fat (34 g), parametrial WAT and subcutaneous WAT weight remained elevated. Paralleling the increase in fat mass, CLA-induced liver enlargement was ameliorated by an increasing intake of dietary fat; in the presence of the 1 g CLA, mice fed 4, 13 and 34 g fat/100 g diet exhibited 190, 100 and 45% greater liver weight, respectively, than mice fed 0 g CLA.

View this table: [in this window] [in a new window]   TABLE 5 Northern blot analysis of parametrial white adipose tissue (WAT) uncoupling protein 2 (UCP2), tumor necrosis factor- (TNF-) and glucose transporter 4 (GLUT4) in mice fed diets containing fat (4 and 34 g/100 g diet) with 0 or 1 g conjugated linoleic acid (CLA)/100 g diet for 5 mo (Experiment 2)1

Mice fed 1 g CLA with 4 g fat exhibited an 81% greater SREBP-1 mRNA and concomitant increases of its target genes, ACC, and SCD-1 but not FAS than mice fed 0 g CLA with 4 g fat, whereas in mice fed 1 g CLA with 34 g fat, these mRNAs did not increase compared with mice fed 0 g CLA with 34 g fat (Table 6). These lipogenic gene profiles were associated with hepatomegaly (Table 3). SREBP-2 mRNA expression and its target genes such as HMG CoA synthetase, HMG CoA reductase and LDL receptor did not differ with and without 1 g CLA/100 g diet in mice fed either 4 or 34 g fat. Expression levels of PPAR and its target genes LPL and ACS also did not increase in the presence of the 1 g CLA. However, the expression level of UCP2, one of the target genes of PPAR, was 114% greater in mice fed 1 g CLA with 4 g fat than in mice fed 0 g CLA with 4 g fat.

View this table: [in this window] [in a new window]   TABLE 6 Northern blot analysis of liver sterol regulatory element-binding protein-1 (SREBP-1), SREBP-2, peroxisome proliferator-activated receptor (PPAR) and their target genes in mice fed diets containing fat (4 and 34 g/100 g diet) with 0 or 1 g conjugated linoleic acid (CLA)/100 g diet for 5 mo (Experiment 2)1, ,2

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

In human studies, 3 g/d CLA was given in a diet containing 70 g/d dietary fat for 2 mo (10) and 4.2 g/d CLA was given in a diet containing 80 g/d dietary fat for 3 mo (12). The former study did not decrease fat mass, but the latter decreased fat mass slightly. Neither study had any adverse effects. The percentage of CLA in dietary fat of these two studies was 4.1 (3/73) and 5.0% (4.2/84.3), respectively. This is a relatively safe range because in mice, 2.5–3.0% CLA in dietary fat did not induce hyperinsulinemia and hepatomegaly, whereas 25% CLA caused a marked hepatomegaly. In contrast, supplementation with 3.4 g of purified trans-10, cis-12 CLA to 58 g/d dietary fat for 3 mo increased insulin resistance and glycemia with a slight reduction of fat mass in abdominally obese men (13). Because the commercially available CLA used in this study contained 36% trans-10, cis-12 CLA, 3.4 g of trans-10, cis-12 CLA is equivalent to 9.4 g commercially available CLA and the percentage of CLA in dietary fat became 13.9% (9.4/67.4). This higher CLA percentage might cause a reduction in fat with hyperinsulinemia and hepatomegaly. In this human study, however, the reduction of fat mass estimated by bioelectrical impedance analysis was 1%; thus, it is unlikely that a marked fat mass reduction occurred. With a high dose of CLA, some functional impairment of adipose tissue function might occur without loss of adipose tissue itself. Indeed, we observed a marked down-regulation of GLUT4 expression in adipose tissues when mice were fed 1 g CLA with 34 g fat (Table 5). Because ablation of GLUT4 in adipose tissues resulted in insulin resistance in skeletal muscles (26), a decrease in GLUT4 in adipose tissues might lead to insulin resistance under some metabolic conditions.

SREBP-1c has been implicated in lipogenic responses to insulin (27). The involvement of SREBP-1c in insulin response is suggested by the finding that SREBP-1c mRNA changes in parallel with blood insulin concentrations (28,29). Hyperinsulinemia observed in mice fed 1 g CLA with 4 g fat might up-regulate SREBP-1c, thus leading to hepatomegaly. Recently, isomer-specific antiobesity effects of CLA were examined. C57BL/6J mice fed dietary trans-10, cis-12 CLA exhibited a decreased WAT mass with hyperinsulinemia and hepatomegaly, whereas WAT mass and liver weight did not differ in mice fed cis-9, trans-11 CLA (30). Ob/ob mice fed trans-10, cis-12 CLA exhibited decreased WAT mass with hyperinsulinemia, whereas ob/ob mice fed cis-9, trans-11 CLA exhibited decreased serum triacylglycerol with a reduction of liver LXR mRNA that led to a decrease in SREBP-1 and lipogenic gene expression (31). Because we used a commercially available mixture of these two types of CLA, a combined effect of these two CLA isomers might be observed in our study. Because dietary trans-10, cis-12 CLA and cis-9, trans-11 CLA had opposite effects on expression of liver SREBP-1, i.e., dietary trans-10, cis-12 CLA up-regulated SREBP-1, whereas cis-9, trans-11 CLA down-regulated SREBP-1, increased expression of FAS was much lower in mice fed commercially available CLA (49% increase in Table 6) than in mice fed trans-10, cis-12 CLA [700% increase, from Fig. 6 in (30)]. The increases in liver lipogenic genes mediated by up-regulation of SREBP-1 support the progression from hyperinsulinemia to hepatomegaly.

Failure to activate PPAR by CLA supplementation might also contribute to hepatomegaly. Up-regulation of liver UCP2 in mice fed 1 g CLA with 4 g fat was not mediated by PPAR activation, although the precise mechanism(s) is not known. This finding is in good agreement with a recent observation that CLA supplementation in PPAR knock-out mice up-regulated liver UCP2 expression (32).

With increasing rates of obesity, the use of nonprescription products for weight loss is likely to increase (33). CLA is one of the promising products in the prevention of lifestyle-related disease. According to this study, a high percentage of CLA in a low fat diet may have adverse effects. To increase dietary fat, adding CLA to the diet as dietary oils may be preferable to the use of capsules. However, for safety and efficacy of the long-term use of these compounds, further human and animal studies are required.

	FOOTNOTES

 
¹ Supported in part by Special Coordination Funds for Promoting Science and Technology from the Japanese Ministry of Education, Culture, Sports, Science and Technology (Tokyo), by research grants from the Japanese Ministry of Health, Labor and Welfare (Tokyo), by a grant from the Promotion of Fundamental Studies in Health Sciences of Organization for Pharmaceutical Safety and Research (OPSR) and by the Uehara Memorial Foundation (Tokyo).

³ Abbreviations used: ACC, acetyl-CoA carboxylase; ACS, acyl-CoA synthetase; BAT, brown adipose tissue; CLA, conjugated linoleic acid; FAS, fatty acid synthase; GLUT4, glucose transporter 4; HMG-CoA, 3-hydroxy-3-methylglutaryl-CoA; LPL, lipoprotein lipase; PCR, polymerase chain reaction; PLSD, protected least significant difference; PPAR, peroxisome proliferator-activated receptor; SCD, stearoyl-CoA desaturase; SREBP, sterol regulatory element-binding protein; T3, 3,5,3'-triiodothyronine; T4, 3,5,3',5'-tetraiodothyronine; UCP, uncoupling protein; WAT, white adipose tissue.

Manuscript received 29 November 2002. Initial review completed 5 January 2003. Revision accepted 26 February 2003.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

1. Chin, S. F., Liu, W., Storkson, J. M., Ha, J. & Pariza, M. W. (1992) Dietary sources of conjugated dienoic isomers of linoleic acid, a newly recognized class of anticarcinogens. J. Food. Compos. Anal. 5:185-197.

2. Pariza, M. W., Park, Y. & Cook, M. E. (2000) Mechanisms of action of conjugated linoleic acid: evidence and speculation. Proc. Soc. Exp. Biol. Med. 223:8-13.[Abstract/Free Full Text]

3. Dugan, M. E. R., Aalhus, J. L., Schaefer, A. L. & Kramer, J. K. G. (1997) The effect of conjugated linoleic acid on fat to lean repartitioning and feed conversion in pigs. Can. J. Anim. Sci. 77:723-725.

4. Park, Y., Albright, K. J., Liu, W., Storkson, J. M., Cook, M. E. & Pariza, M. W. (1997) Effect of conjugated linoleic acid on body composition in mice. Lipids 32:853-858.[Medline]

5. Belury, M. A. & Kempa-Steczko, A. (1997) Conjugated linoleic acid modulates hepatic lipid composition in mice. Lipids 32:199-204.[Medline]

6. Deckere, E. A. M., Amelsvoort, J. M. M., McNeill, G. P. & Jones, P. (1999) Effects of conjugated linoleic acid (CLA) isomers on lipid levels and peroxisome proliferation in the hamster. Br. J. Nutr. 82:309-317.[Medline]

7. Tsuboyama-Kasaoka, N., Takahashi, M., Tanemura, K., Kim, H. J., Tange, T., Okuyama, H., Kasai, M., Ikemoto, S. & Ezaki, O. (2000) Conjugated linoleic acid supplementation reduces adipose tissue by apoptosis and develops lipodystrophy in mice. Diabetes 49:1534-1542.[Abstract]

8. Lawrence, R. D. (1946) Lipodystrophy and hepatomegaly with diabetes, lipaemia, and other metabolic disturbances. Lancet 1:724-731.

9. Seip, M. & Trygstad, O. (1996) Generalized lipodystrophy, congenital and acquired (lipoatrophy). Acta Paediatr. Suppl. 413:2-28.[Medline]

10. Zambell, K. L., Keim, N. L., Van, , Loan, M. D., Gale, B., Benito, P., Kelley, D. S. & Nelson, G. J. (2000) Conjugated linoleic acid supplementation in humans: effects on body composition and energy expenditure. Lipids 35:777-782.[Medline]

11. Blankson, H., Stakkestad, J. A., Fagertun, H., Thom, E., Wadstein, J. & Gudmundsen, O. (2000) Conjugated linoleic acid reduces body fat mass in overweight and obese humans. J. Nutr. 130:2943-2948.[Abstract/Free Full Text]

11. Smedman, A. & Vessby, B. (2001) Conjugated linoleic acid supplementation in humans—metabolic effects. Lipids 36:773-781.[Medline]

13. Riserus, U., Arner, P., Brismar, K. & Vessby, B. (2002) Treatment with dietary trans10cis12 conjugated linoleic acid causes isomer-specific insulin resistance in obese men with the metabolic syndrome. Diabetes Care 25:1516-1521.[Abstract/Free Full Text]

14. Delany, J. P., Blohm, F., Truett, A. A., Scimeca, J. A. & West, D. B. (1999) Conjugated linoleic acid rapidly reduces body fat content in mice without affecting energy intake. Am. J. Physiol. 276:R1172-R1179.

15. Ohnuki, K., Haramizu, S., Ishihara, K. & Fushiki, T. (2001) Increased energy metabolism and suppressed body fat accumulation in mice by a low concentration of conjugated linoleic acid. Biosci. Biotechnol. Biochem. 65:2200-2204.[Medline]

16. Reeves, P. G., Nielsen, F. H. & Fahey, G. C., Jr (1993) AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN-76A rodent diet. J. Nutr. 123:1939-1951.

17. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J. & Rutter, W. J. (1979) Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18:5294-5299.[Medline]

18. Horton, J. D., Goldstein, J. L. & Brown, M. S. (2002) SREBPs: activators of the complete program of cholesterol and fatty acid synthesis in the liver. J. Clin. Investig. 109:1125-1131.[Medline]

19. Schoonjans, K., Staels, B. & Auwerx, J. (1996) The peroxisome proliferator activated receptors (PPARS) and their effects on lipid metabolism and adipocyte differentiation. Biochim. Biophys. Acta 1302:93-109.[Medline]

20. Nakatani, T., Tsuboyama-Kasaoka, N., Takahashi, M., Miura, S. & Ezaki, O. (2002) Mechanism for peroxisome proliferator-activated receptor-alpha activator-induced up-regulation of UCP2 mRNA in rodent hepatocytes. J. Biol. Chem. 277:9562-9569.[Abstract/Free Full Text]

21. Tsuboyama-Kasaoka, N., Tsunoda, N., Maruyama, K., Takahashi, M., Kim, H., Ikemoto, S. & Ezaki, O. (1998) Up-regulation of uncoupling protein 3 (UCP3) mRNA by exercise training and down-regulation of UCP3 by denervation in skeletal muscles. Biochem. Biophys. Res. Commun. 247:498-503.[Medline]

22. Kim, H. -J., Takahashi, M. & Ezaki, O. (1999) Fish oil feeding decreases mature sterol regulatory element-binding protein 1 (SREBP-1) by down-regulation of SREBP-1c mRNA in mouse liver. J. Biol. Chem. 274:25892-25898.[Abstract/Free Full Text]

23. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual 2nd ed. 1989 Cold Spring Harbor Laboratory Cold Spring Harbor, NY.

24. Takahashi, M., Ikemoto, S. & Ezaki, O. (1999) Effect of the fat/carbohydrate ratio in the diet on obesity and oral glucose tolerance in C57BL/6J mice. J. Nutr. Sci. Vitaminol. 45:583-593.

25. Belury, M. A. (2002) Dietary conjugated linoleic acid in health: physiological effects and mechanisms of action. Annu. Rev. Nutr. 22:505-531.[Medline]

26. Abel, E. D., Peroni, O., Kim, J. K., Kim, Y. B., Boss, O., Hadro, E., Minnemann, T., Shulman, G. I. & Kahn, B. B. (2001) Adipose-selective targeting of the GLUT4 gene impairs insulin action in muscle and liver. Nature (Lond.) 409:729-733.[Medline]

27. Tobin, K. A. R., Ulven, S. M., Schuster, G. U., Steineger, H. H., Andresen, S. M., Gustafsson, J.-A. & Nebb, H. I. (2002) Liver X receptors as insulin-mediating factors in fatty acid and cholesterol biosynthesis. J. Biol. Chem. 277:10691-10697.[Abstract/Free Full Text]

28. Shimomura, I., Bashmakov, Y., Ikemoto, S., Horton, J. D., Brown, M. S. & Goldstein, J. L. (1999) Insulin selectively increases SREBP-1c mRNA in the livers of rats with streptozotocin-induced diabetes. Proc. Natl. Acad. Sci. U.S.A. 96:13656-13661.[Abstract/Free Full Text]

29. Foretz, M., Guichard, C., Ferre, P. & Foufelle, F. (1999) Sterol regulatory element binding protein-1c is a major mediator of insulin action on the hepatic expression of glucokinase and lipogenesis-related genes. Proc. Natl. Acad. Sci. U.S.A. 96:14191-14192.[Free Full Text]

30. Clement, L., Poirier, H., Niot, I., Bocher, V., Guerre-Millo, M., Krief, S., Staels, B. & Besnard, P. (2002) Dietary trans-10, cis-12 conjugated linoleic acid induces hyperinsulinemia and fatty liver in the mouse. J. Lipid Res. 43:1400-1409.[Abstract/Free Full Text]

31. Roche, H. M., Noone, E., Sewter, C., Mc, , Bennett, S., Savage, D., Gibney, M. J., O’Rahilly, S. & Vidal-Puig, A. J. (2002) Isomer-dependent metabolic effects of conjugated linoleic acid: insights from molecular markers sterol regulatory element-binding protein-1c and LXR. Diabetes 51:2037-2044.[Abstract/Free Full Text]

32. Peters, J. M., Park, Y., Gonzalez, F. J. & Pariza, M. W. (2001) Influence of conjugated linoleic acid on body composition and target gene expression in peroxisome proliferator-activated receptor alpha-null mice. Biochim. Biophys. Acta 1533:233-242.[Medline]

33. Blanck, H. M., Khan, L. K. & Serdula, M. K. (2001) Use of nonprescription weight loss products; results from a multistate survey. J. Am. Med. Assoc. 286:930-935.[Abstract/Free Full Text]

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