Increasing the Amount of Fat in a Conjugated Linoleic AcidSupplemented

8/11/2019 Increasing the Amount of Fat in a Conjugated Linoleic AcidSupplemented

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Nutrient-Gene Interactions

Increasing the Amount of Fat in a Conjugated Linoleic Acid–SupplementedDiet Reduces Lipodystrophy in Mice1

Nobuyo Tsuboyama-Kasaoka, Hiromi Miyazaki, Seiichi Kasaoka* and Osamu Ezaki2

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

ABSTRACT Conjugated linoleic acid (CLA) is a naturally occurring group of dienoic derivatives of linoleic acidfound 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/100g 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 tissuewas 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 gCLA, subcutaneous white adipose tissue (WAT) weight was 48% lower than in mice fed 0 g CLA. The mice fed 0.1 gCLA did not exhibit hepatomegaly and insulin resistance. In Experiment 2, mice were fed for 5 mo different amountsof 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 gCLA 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 ofCLA-mediated lipodystrophy. J. Nutr. 133: 1793–1799, 2003.

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

Conjugated linoleic acid (CLA)3 is a group of positionaland 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 considerableattention because of its anticarcinogenic and antiatherogenicproperties and its ability to reduce body fat while enhancinglean body mass (2). Reduction of body fat by CLA was ob-served 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 ina low fat diet, a marked reduction of fat mass by CLA resultedin insulin resistance and hepatomegaly (7). These metabolicabnormalities are characteristics of lipodystrophy (8,9). Thedecrease in fat tissue was due to apoptosis. Tumor necrosis

factor- (TNF-) and uncoupling protein 2 (UCP2) mRNAslevels were 11- and 5-fold greater, respectively, in isolatedadipocytes from mice fed CLA than from those not fed CLA.

Because TNF- induces apoptosis of adipocytes and up-regu-lates UCP2 mRNA in mice, a marked increase of TNF-mRNA with an increase of UCP2 might cause CLA-inducedapoptosis (7). Because continuous leptin infusion reverses hy-perinsulinemia and partly prevents lipid accumulation in livertissues, hepatomegaly was likely due in part to hypoleptinemiaand hyperinsulinemia (7). We hypothesized that the markedreduction of fat mass caused hyperinsulinemia and hepato-megaly.

In human studies, 3 g/d of CLA supplementation for 2 modid not affect body composition and energy expenditure innormal weight women (10); 3.4 g/d CLA supplementation for3 mo reduced body fat mass in overweight and obese subjects(11); and 4.2 g/d CLA supplementation reduced body fat inmen (12). These three trials did not have any overt adverseeffects. These data are in agreement with our hypothesis; whenwe assumed that a marked decrease of fat mass contributed toCLA-induced hyperinsulinemia and hepatomegaly, the slightdecrease or no change in body fat observed in human studymight not have adverse effects. In contrast, in a recent ran-domized, double-blind controlled trial, supplementation with3.4 g of purified trans-10, cis-12 CLA for 3 mo increased insulinresistance and glycemia with a slight reduction of fat mass inabdominally obese men. When a mixture of CLA (trans-10,

1 Supported in part by Special Coordination Funds for Promoting Science and

Technology from the Japanese Ministry of Education, Culture, Sports, Scienceand Technology (Tokyo), by research grants from the Japanese Ministry of Health,Labor and Welfare (Tokyo), by a grant from the Promotion of Fundamental Studiesin Health Sciences of Organization for Pharmaceutical Safety and Research(OPSR) and by the Uehara Memorial Foundation (Tokyo).

2 To whom correspondence should be addressed. E-mail: [email protected] Abbreviations used: ACC, acetyl-CoA carboxylase; ACS, acyl-CoA syn-

thetase; BAT, brown adipose tissue; CLA, conjugated linoleic acid; FAS, fatty acidsynthase; GLUT4, glucose transporter 4; HMG-CoA, 3-hydroxy-3-methylglutaryl-CoA; LPL, lipoprotein lipase; PCR, polymerase chain reaction; PLSD, protectedleast significant difference; PPAR, peroxisome proliferator-activated receptor;SCD, stearoyl-CoA desaturase; SREBP, sterol regulatory element-binding pro-tein; T3, 3,5,3-triiodothyronine; T4, 3,5,3,5-tetraiodothyronine; UCP, uncou-pling protein; WAT, white adipose tissue.

0022-3166/03 $3.00 © 2003 American Society for Nutritional Sciences.Manuscript received 29 November 2002. Initial review completed 5 January 2003. Revision accepted 26 February 2003.

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cis-12 CLA and cis-9, trans-11 CLA) was given, however, noadverse effects were observed (13). In contrast to our hypoth-esis, these data indicated that trans-10, cis-12 CLA per secauses insulin resistance in the absence of a marked fat massdecrease.

To examine in greater detail the contribution of fat massdecrease by CLA supplementation to hyperinsulinemia andhepatomegaly, we examined the effects of CLA under twodietary conditions, i.e., a decreased amount of CLA and anincreased amount of dietary fat. If we assume that a markeddecrease in fat mass was a major cause of hypoleptinemia, a

reduction in the amount of CLA might simply decrease fatmass moderately but not cause the adverse effects. At present,several dose-dependent studies of CLA in mice have beenconducted, and a minimal effective dose of CLA to reducebody fat can be estimated. Belury et al. (5) showed that micefed 0.5 g CLA/100 g diet in a low fat diet for 6 wk had reducedbody weight but increased liver triglyceride concentration.DeLany et al. (14) reported that male AKR/J mice fed a highfat diet containing 0.5 g CLA/100 g diet for 12 wk exhibitedreduced body fat but increased plasma insulin concentration.Ohnuki et al. (15) showed that Std ddY male mice fed a lowfat diet containing 0.25 g CLA/100 g diet for 4 wk exhibitedreduced 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 evaluatedby an insulin tolerance test or glucose clamp studies. Anotherway in which to increase adipose tissue mass is to increasedietary fat intake. We increased dietary fat while the dose of CLA was kept stable. Through these two dietary interven-tions, we estimated the role of a decrease in adipose tissue onCLA-induced hyperinsulinemia and hepatomegaly.

MATERIALS AND METHODS

Mice. Female C57BL/6J mice were obtained from Tokyo Labo-ratory Animals Science (Tokyo, Japan) at 7 wk of age and fed anormal laboratory diet (CE2, Clea, Tokyo, Japan) for 1 wk to stabilizeall metabolic conditions. Each cage contained 5– 6 mice. Mice wereexposed to 12-h light:dark cycle and maintained at a constant tem-perature of 22°C. All procedures were in accordance with the Na-

tional Institute of Health and Nutrition Guidelines for the Care andUse of Laboratory Animals in Japan.

Diets. The dietary compositions used in our experiments areshown in Table 1. Saf flower oil was the source of dietary fat. Saf floweroil (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 providedand prepared as a free fatty acid by Rinoru Oil Mills (Tokyo, Japan)and stored frozen in plastic bottles blanketed with nitrogen. Linoleicacid 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 adough with the addition of water, rolled into pellets, wrapped with

Saran wrap (Asahikasei, Tokyo, Japan) and stored at 20°C until useto minimize fatty acid oxidation.Preliminary feeding trials were conducted and the composition of

each diet was adjusted so that the daily intake of energy and thecomposition of each diet except fat and carbohydrate were nearlyidentical. Fresh food was provided to the mice biweekly. The mate-rials and methods of diet preparation were the same as those used inour previous studies (7).

Experiment 1: dose effects of CLA. Mice were fed different dosesof CLA (0, 0.1 and 1 g CLA/100 g diet) in a fixed 4 g fat/100 g dietfor 5 mo. The composition of each diet is shown in Table 1. Theexperiment was conducted twice. For the insulin tolerance test, 9 wkafter CLA supplementation, human insulin (Humulin R, Eli Lilly Japan K.K., Kobe, Japan) was injected intraperitoneally (4.5 pmol/gbody) into fed mice. Blood glucose was measured in samples obtained

from the tail tip before and 15, 30, 60, 90 and 120 min after insulininjection, 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 absorpti-ometry (Lunar PIXI mus2 densitometer, Lunar Corporation, Madi-son, WI) during wk 19 of feeding. At the end of the experiments, fedmice were anesthetized at 1000 h by intraperitoneal injection of pentobarbital sodium (0.08 mg/g body, Nembutal, Abbot, NorthChicago, IL).

Experiment 2: effects of dietary fat amount on CLA-inducedlipodystrophy. Mice were fed different amounts of dietary fat (4, 13and 34 g fat/100 g diet) in 0 or 1 g CLA/100 g diet for 5 mo. Thecomposition of each diet is shown in Table 1. The experiment wasconducted twice. Energy intake was measured during 13–14 wk of feeding. Blood samples were obtained by cutting the tail end. Infood-deprived mice, immunoreactive insulin was measured at 15 wkby an Insulin Assay Kit (Morinaga, Kanagawa, Japan). Plasma leptinwas measured in fed mice at 19 wk by a Mouse Leptin Assay Kit

TABLE 1

Composition of the experimental diets containing fat (4, 13, and 34 g/100 g diet) with or without conjugated linoleic acid (CLA) 1

Fat, g/100 g diet 4 13 34

CLA, g/100 g diet 0 0.1 1 0 1 0 1

g/kg diet

CLA mixture2 0 1.4 14.0 0 14.0 0 14.0Safflower oil 40.0 38.6 26.0 130.0 116.0 335.0 321.0Casein 200.0 200.0 200.0 228.0 228.0 290.0 290.0Sucrose 110.3 110.3 110.3 125.8 125.8 232.9 232.9Cornstarch 551.7 551.7 551.7 404.5 404.5 0.0 0.0

Vitamin mix 10.0 10.0 10.0 11.4 11.4 14.5 14.5Mineral mix 35.0 35.0 35.0 39.9 39.9 50.8 50.8Cellulose powder 50.0 50.0 50.0 57.0 57.0 72.4 72.4L-Cystine 3.0 3.0 3.0 3.4 3.4 4.4 4.4

Fat energy, % of total energy 10.5 10.5 10.5 30.0 30.0 60.5 60.5CLA, % of total fat 0 2.5 25 0 7.7 0 3.0

1 Casein, sucrose, dextrinized cornstarch, vitamin and mineral mixture (AIN-93G) (16), and cellulose powder were purchased from oriental Yeast(Tokyo, Japan), L-cystine from Wako (Osaka, Japan) and safflower oil from Benibana (Tokyo, Japan).

2 Because 72.2 g/100 g of a CLA mixture is pure CLA, more of the mixture was added to obtain the net amount.

TSUBOYAMA-KASAOKA ET AL.1794



(Morinaga), 3,5,3-triiodothyronine (T3) by T-3 RIA Bead (Dain-abot, Tokyo, Japan) and 3,5,3,5-tetraiodothyronine (T4) by T-4RIA Bead (Dainabot). At the end of the experiments, fed mice wereanesthetized at1000 h by intraperitoneal injection of pentobarbitalsodium (0.08 mg/g body, Nembutal, Abbot). Liver, gastrocnemius,and parametrial white adipose tissue (WAT) were isolated immedi-ately, weighed and homogenized in guanidine-thiocyanate; RNA wasprepared by the method of Chirgwin et al. (17). Gene expressionanalysis was performed in mice fed 4 and 34 g fat/100 g diet.

Preparation of cDNA probe and Northern blot. To study themechanism(s) of CLA-induced hepatomegaly, we examined expres-sion levels of several target genes of sterol regulatory element-bindingprotein-1 (SREBP-1), SREBP-2 and peroxisome proliferator-activa-tor receptor (PPAR). The target genes of SREBP-1c involved infatty 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 areregulated 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 UCP2were 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 wereobtained by PCR from first strand cDNA using mouse liver totalRNA as described previously (22). The cDNA probes for rat LDLreceptor and ACS were kindly provided by Dr. T. Yamamoto atTohoku University, rat ACC and FAS by Dr. N. Iritani at Te-zukayama Gakuin College, PPAR by Dr. T. Osumi at Himeji Insti-tute of Technology, and mouse SCD-1 and glucose transporter 4(GLUT4) by Dr. Daniel M. Lane at Johns Hopkins University. ThesecDNAs were used as probes for Northern blots. A portion of RNA(15 g per lane) was denatured with glyoxal and dimethyl sulfoxideand analyzed by electrophoresis in 1 g/100 g agarose gels. Aftertransfer to nylon membranes (NEN, Boston, MA) and UV crosslink-ing, RNA blots were stained with methylene blue to locate 28S and18S rRNAs and to ascertain the amount of loaded RNAs (23). Theblots were hybridized overnight at 42°C with cDNAs, which had

been labeled with 32P-dCTP (NEN) by a Megaprime DNA labelingkit (Amersham Pharmacia Biotech, Buckinghamshire, England). Thefilters were washed several times with 1 X SSC, 0.1 g/100 g of SDS atroom temperature, washed twice at 50°C and then exposed to X-rayfilm at 80°C. The amounts of each mRNA were quantitated withan image analyzer (BAS 2000, Fuji Film, Tokyo, Japan).

Statistical methods. Data were analyzed by one-way (Experi-ment 1) or two-way (Experiment 2) ANOVA, and significant differ-ences among means were identified by Fisher’s protected least signif-icant difference (PLSD) test (Super ANOVA, Abacus Concepts,Berkeley, CA). When the variances were not homogeneous accord-ing to Bartlett’s test (Statview 5.0, Abacus Concepts), data weretransformed logarithmically, and then the transformed data wereanalyzed by ANOVA followed by multiple comparison. When thevariances were not homogenous even after logarithmic transforma-tion, 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 curveof each group in Figure 1 was compared by repeated-measuresANOVA, followed by Fisher’s PLSD test (Statview 5.0). Differenceswere considered significant at P 0.05. Values in the text are means SEM.

RESULTS

Dose effects of CLA (Experiment 1). Energy intake andbody weight did not differ among mice fed 0, 0.1 and 1 gCLA/100 g in a fixed amount of fat (4 g/100 g diet) (data notshown). As observed in our previous study (7), mice fed 1 gCLA, retroperitoneal and subcutaneous WAT and brown ad-ipose 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). Inmice 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 notexhibit hepatomegaly and insulin resistance. The decrease inwhole-body fat mass was also assessed by dual energy X-rayabsorptiometry (Table 2). However, total fat in mice fed 0.1 gCLA did not differ from that in mice fed 0 g CLA, whereastotal fat in mice fed 1 g CLA was 53% lower than that in micefed 0 g CLA. Importantly, mice fed 0.1 g CLA did not exhibithepatomegaly, whereas mice fed 1 g CLA did. Furthermore,the insulin-mediated glucose lowering effect was not impairedin mice fed 0.1 g CLA, whereas this effect was markedlyimpaired in mice fed 1 g CLA (P 0.0001) ( Fig. 1). Thus, a

lower dose of CLA can effectively reduce body fat mass with-out the adverse effects after 5 mo of feeding.

FIGURE 1 Insulin tolerance tests of mice fed diets containing 4 gfat/100 g diet with 0, 0.1 or 1 g conjugated linoleic acid (CLA)/100 g dietfor 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.

TABLE 2

Body fat, white adipose tissue (WAT), brown adipose tissue

(BAT), liver and spleen weights in 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 5 mo (Experiment 1)1

CLA, g/100 g diet 0 0.1 1

Body fat, g/100 g body weight 25.8 2.7b 22.3 2.6b 12.0 0.6a

g

Parametrial WAT 0.70 0.12b 0.58 0.11b 0.19 0.03a

Retroperitoneal WAT 0.15 0.03b 0.09 0.02b 0.00 0.00a

Subcutaneous WAT 0.81 0.12c 0.42 0.08b 0.04 0.00a

BAT 0.10 0.01b 0.12 0.02b 0.01 0.00a

Liver 1.07 0.07a 1.08 0.07a 2.97 0.19b

Spleen 0.09 0.01a 0.08 0.01a 0.19 0.03b

1

Values are means SEM

, n

5–10. Data were transformed log-arithmically before analysis, and then transformed data were analyzedby ANOVA followed by Fisher’s protected least significant differencetest. Means in a row without a common letter differ, P 0.05.

CONJUGATED LINOLEIC ACID, FAT AND LIPODYSTROPHY 1795



Effects of dietary fat amount on CLA-induced lipodystrophy (Experiment 2). In agreement with our previous ob-servations (24), when dietary fat was increased from 4 to34 g fat/100 g diet without CLA supplementation, miceexhibited increased parametrial, retroperitoneal and subcu-taneous WAT weights in a dose-dependent manner (Table3). As observed in our previous study with 4 g fat/100 g diet(7), mice fed 1 g CLA exhibited ablated retroperitoneal andsubcutaneous WAT, decreased parametrial WAT and in-creased liver and spleen weights. However, mice fed in-creased dietary fat with or without CLA supplementationexhibited increased WAT weight. In mice fed 1 g CLA with

the highest amount of fat (34 g), parametrial WAT andsubcutaneous WAT weight remained elevated. Parallelingthe increase in fat mass, CLA-induced liver enlargementwas ameliorated by an increasing intake of dietary fat; in thepresence of the 1 g CLA, mice fed 4, 13 and 34 g fat/100 gdiet exhibited 190, 100 and 45% greater liver weight,respectively, than mice fed 0 g CLA.

Mice fed 1 g CLA with 4 g fat exhibited 236, 104 and 81%greater concentrations of plasma insulin, T3 and T4, respec-tively, but decreased plasma leptin concentration (79%) thanmice fed the 0 g CLA with 4 g fat (Table 4). However, micefed 1 g CLA with 34 g fat did not differ in plasma insulin,

leptin, and T3 and T4 concentrations, compared with mice fed0 g CLA with 34 g fat. Thus, normal leptin, insulin, and T3and T4 concentrations and the absence of a marked hepato-megaly in mice fed 1 g CLA with 34 g fat support the idea thathormonal imbalance may be closely related to the develop-ment of hepatomegaly. The 100% increases in thyroid hor-mone concentrations in mice fed 1 g CLA with 4 g fat mayalso contribute to fat mass decrease because of increased ther-mogenesis due to CLA supplementation (15).

As described in our previous study (7), mice fed 1 g CLAwith 4 g fat exhibited markedly increased UCP2 and TNF-,and decreased GLUT4 mRNA expressions in WAT, whereas

mice fed 1 g with 34 g fat did not exhibit increases in UCP2and TNF- mRNA (Table 5). However, in mice fed 1 g CLAwith 34 g fat, GLUT4 mRNA in adipose tissues was 63% lowerthan in mice fed 0 g CLA with 34 g fat. In contrast, ingastrocnemius, mice fed 1 g CLA with 4 and 34 g fat had 50and 20% increases in GLUT4 expression, respectively (datanot shown).

Liver gene expression (Experiment 2). Mice fed 1 g CLAwith 4 g fat exhibited an 81% greater SREBP-1 mRNA andconcomitant increases of its target genes, ACC, and SCD-1but not FAS than mice fed 0 g CLA with 4 g fat, whereas inmice fed 1 g CLA with 34 g fat, these mRNAs did not increase

TABLE 3

Body weight, white adipose tissue (WAT), brown adipose tissue (BAT), liver and spleen weights in mice fed diets containing fat (4,

13 and 34 g/100 g diet) with 0 or 1 g conjugated linoleic acid (CLA)/100 g diet for 5 mo (Experiment 2) 1

Fat, g/100 g diet 4 13 34 ANOVA P2

CLA, g/100 g diet 0 1 0 1 0 1 Fat CLA Fat CLA

g

Body weight 24.0 1.3a 21.5 0.8a 27.8 1.4b 23.0 0.5a 32.5 1.8c 23.9 1.4a 0.001 0.001 NSParametrial WAT3 0.64 (0.23–1.19)b 0.19 (0.04–0.32)a 1.28 (0.70–1.69)bc 0.66(0.55–0.77)b 1.68 (1.06–1.82)c 0.77 (0.68–1.57)bc

RetroperitonealWAT 0.15 0.02c 0.00 0.00a 0.27 0.04d 0.02 0.00b 0.34 0.02d 0.08 0.01c 0.001 0.001 0.001

SubcutaneousWAT 0.83 0.11d 0.04 0.00a 1.38 0.26e 0.07 0.01b 2.29 0.12e 0.48 0.08c 0.001 0.001 0.001

BAT 0.10 0.01b 0.01 0.00a 0.14 0.02c 0.03 0.00a 0.11 0.01bc 0.08 0.01b 0.001 0.001 0.001Liver 1.03 0.08ab 2.99 0.17d 0.95 0.03a 1.90 0.09c 0.89 0.10a 1.29 0.15b 0.001 0.001 0.001Spleen 0.09 0.01ab 0.19 0.03c 0.09 0.01ab 0.12 0.01b 0.06 0.01a 0.09 0.01ab 0.01 0.001 NS

1 Values are means SEM, n 4 –11. Means in a row without a common letter differ, P 0.05.2 P-values from two-way ANOVA; NS, P 0.05.3 Results are medians with ranges below in parentheses. The effects of dietary treatment were examined by Kruskal-Wallis ANOVA followed by

Kolmogorov-Smirnov two-sample test.

TABLE 4

Plasma insulin, leptin, 3,5,3-triiodothyronine (T3) and 3,5,3 ,5-tetraiodothyronine (T4) concentrations in mice fed the 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

Fat, g/100 g diet 4 34 ANOVA P2

CLA, g/100 g diet 0 1 0 1 Fat effect CLA effect Fat CLA

Insulin, nmol/L plasma 0.11 0.01a 0.37 0.07b 0.07 0.01a 0.11 0.02a 0.001 0.001 0.05Leptin,3 g/L plasma 6.8 (5.5–8.5)b 1.4 (0.5–2.1)a 25.5 (25.0–26.0)c 16.0 (11.3–26.0)c

T3, nmol/L plasma 0.48 0.02a 0.98 0.05b 0.46 0.02a 0.44 0.04a 0.001 0.001 0.001T4, nmol/L plasma 4.8 0.4b 8.7 0.5c 4.1 0.2ab 3.4 0.4a 0.001 0.001 0.001

1 Values are means SEM, n 3– 4. Means in a row without a common letter differ, P 0.05.2

P-values from two-way ANOVA.3 Results are medians with ranges in parentheses. The effects of dietary treatment were examined by Kruskal-Wallis ANOVA followed byKolmogorov-Smirnov two-sample test.




compared with mice fed 0 g CLA with 34 g fat ( Table 6).These lipogenic gene profiles were associated with hepatomeg-aly (Table 3). SREBP-2 mRNA expression and its target genes

such as HMG CoA synthetase, HMG CoA reductase and LDLreceptor did not differ with and without 1 g CLA/100 g diet inmice fed either 4 or 34 g fat. Expression levels of PPAR andits target genes LPL and ACS also did not increase in thepresence of the 1 g CLA. However, the expression level of UCP2, one of the target genes of PPAR, was 114% greater inmice fed 1 g CLA with 4 g fat than in mice fed 0 g CLA with4 g fat.

DISCUSSION

Supplementation of CLA causes a variety of effects. CLAhas shown anticarcinogenic, antiatherogenic and antiobesity

effects (25). However, when 1 g CLA/100 g diet in a low fatdiet (4 g fat/100 g diet) was given to mice, a marked reductionof fat mass by CLA resulted in lipodystrophy (insulin resis-

tance and hepatomegaly) (7). A marked reduction in fat massby apoptosis resulted in hypoleptinemia, which may lead toinsulin resistance and hepatomegaly. Thus, we speculated thata small but not a marked reduction of fat mass might not causehyperinsulinemia and hepatomegaly. Evidence in this studythat mice fed a diet containing either 0.1 g CLA with 4 g fator 1 g CLA with 34 g fat/100 g exhibited moderately reducedfat mass but no hyperinsulinemia and hepatomegaly supportsthis hypothesis. CLA was present in dietary fat at 2.5 and 3.0%when the diet contained 0.1 g CLA with 4 g fat and 1 g CLAwith 34 g fat, respectively, whereas it was present at 25% whenthe diet contained 1 g CLA with 4 g fat (Table 1). Thus, a

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



%

UCP2 100 8a 482 17c 167 32b 128 17ab 0.001 0.001 0.001TNF- 100 31a 828 79c 296 55b 306 82b 0.05 0.001 0.001GLUT4 100 13c 9 1a 38 5b 14 3a NS 0.001 0.01

1 Values are means SEM, n 3– 4. The data are shown as relative value to mRNA levels of 4 g fat/100 g diet, 0 g CLA/100 g diet fed mice. Meansin a row without a common letter differ, P 0.05.

2 P-values from two-way ANOVA; NS, P 0.05.

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



%

SREBP-1 and its target genesSREBP-1 100 15a 181 24b 138 15ab 173 15b NS 0.01 NS

ACC 100

22a

275

11b

108

11a

128

7a

0.001

0.001

0.001SCD 1 100 15b 178 5c 21 4a 41 4a 0.001 0.001 0.01FAS 100 21bc 149 21c 42 5a 67 11ab 0.001 0.05 NS

SREBP-2 and its target genesSREBP-2 100 8 102 8 116 4 121 8 0.05 NS NSHMG-CoA synthase 100 14a 101 16a 202 36b 169 25ab 0.01 NS NSHMG-CoA reductase 100 10 174 24 166 19 159 25 NS NS NSLDL receptor 100 8 134 10 98 7 88 18 NS NS NS

PPAR and its target genesPPAR 100 6 142 12 162 17 184 34 0.05 NS NSLPL 100 20 101 23 64 7 47 13 0.05 NS NS

ACS 100 11b 57 10a 128 10b 100 9b 0.01 0.01 NSUCP2 100 8a 214 14b 156 23ab 151 23ab NS 0.05 0.05

1 Values are means SEM, n 3– 4. The data are shown as relative value to mRNA levels of 4 g fat/100 g diet, 0 g CLA/100 g diet fed mice. Meansin a row without a common letter differ, P 0.05.

2

Abbreviations: ACC, acetyl-CoA carboxylase; SCD-1, stearoyl-CoA desaturase-1; FAS, fatty acid synthase; HMG, 3-hydroxy-3-methylglutaryl;LPL, lipoprotein lipase; ACS, acyl-CoA synthetase; UCP, uncoupling protein.3 P-values from two-way ANOVA; NS, P 0.05.




lower percentage of CLA in dietary fat resulted in a mildreduction of fat mass without adverse effects, whereas a higherpercentage resulted in a marked reduction of fat mass withlipodystrophy.

In human studies, 3 g/d CLA was given in a diet containing70 g/d dietary fat for 2 mo (10) and 4.2 g/d CLA was given ina diet containing 80 g/d dietary fat for 3 mo (12). The formerstudy did not decrease fat mass, but the latter decreased fat

mass slightly. Neither study had any adverse effects. Thepercentage 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 relativelysafe range because in mice, 2.5–3.0% CLA in dietary fat didnot induce hyperinsulinemia and hepatomegaly, whereas 25%CLA caused a marked hepatomegaly. In contrast, supplemen-tation with 3.4 g of purified trans-10, cis-12 CLA to 58 g/ddietary fat for 3 mo increased insulin resistance and glycemiawith a slight reduction of fat mass in abdominally obese men(13). Because the commercially available CLA used in thisstudy contained 36% trans-10, cis-12 CLA, 3.4 g of trans-10,cis-12 CLA is equivalent to 9.4 g commercially available CLAand the percentage of CLA in dietary fat became 13.9%(9.4/67.4). This higher CLA percentage might cause a reduc-

tion in fat with hyperinsulinemia and hepatomegaly. In thishuman study, however, the reduction of fat mass estimated bybioelectrical impedance analysis was 1%; thus, it is unlikelythat a marked fat mass reduction occurred. With a high doseof CLA, some functional impairment of adipose tissue func-tion might occur without loss of adipose tissue itself. Indeed,we observed a marked down-regulation of GLUT4 expressionin adipose tissues when mice were fed 1 g CLA with 34 g fat(Table 5). Because ablation of GLUT4 in adipose tissuesresulted in insulin resistance in skeletal muscles (26), a de-crease in GLUT4 in adipose tissues might lead to insulinresistance under some metabolic conditions.

SREBP-1c has been implicated in lipogenic responses toinsulin (27). The involvement of SREBP-1c in insulin re-

sponse is suggested by the finding that SREBP-1c mRNAchanges in parallel with blood insulin concentrations (28,29).Hyperinsulinemia observed in mice fed 1 g CLA with 4 g fatmight up-regulate SREBP-1c, thus leading to hepatomegaly.Recently, isomer-specific antiobesity effects of CLA were ex-amined. C57BL/6J mice fed dietary trans-10, cis-12 CLA ex-hibited a decreased WAT mass with hyperinsulinemia andhepatomegaly, whereas WAT mass and liver weight did notdiffer in mice fed cis-9, trans-11 CLA (30). Ob/ob mice fedtrans-10, cis-12 CLA exhibited decreased WAT mass withhyperinsulinemia, whereas ob/ob mice fed cis-9, trans-11 CLAexhibited decreased serum triacylglycerol with a reduction of liver LXR mRNA that led to a decrease in SREBP-1 andlipogenic gene expression (31). Because we used a commer-

cially available mixture of these two types of CLA, a combinedeffect of these two CLA isomers might be observed in ourstudy. Because dietary trans-10, cis-12 CLA and cis-9, trans-11CLA had opposite effects on expression of liver SREBP-1, i.e.,dietary trans-10, cis-12 CLA up-regulated SREBP-1, whereascis-9, trans-11 CLA down-regulated SREBP-1, increased ex-pression of FAS was much lower in mice fed commerciallyavailable CLA (49% increase in Table 6) than in mice fedtrans-10, cis-12 CLA [700% increase, from Fig. 6 in (30)]. Theincreases in liver lipogenic genes mediated by up-regulation of SREBP-1 support the progression from hyperinsulinemia tohepatomegaly.

Failure to activate PPAR by CLA supplementation mightalso 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 observationthat CLA supplementation in PPAR knock-out mice up-regulated liver UCP2 expression (32).

With increasing rates of obesity, the use of nonprescriptionproducts for weight loss is likely to increase (33). CLA is oneof the promising products in the prevention of lifestyle-relateddisease. According to this study, a high percentage of CLA ina low fat diet may have adverse effects. To increase dietary fat,

adding CLA to the diet as dietary oils may be preferable to theuse of capsules. However, for safety and ef ficacy of the long-term use of these compounds, further human and animalstudies are required.

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Increasing the Amount of Fat in a Conjugated Linoleic AcidSupplemented