ABSTRACT: More than half of the U.S. population has a bodymass index of 25 kg/m2 or more, which classifies them as over-weight or obese. Obesity is often associated with comorbiditiessuch as diabetes, cardiovascular diseases, and cancer. CLA andchromium have emerged as major dietary supplements that re-duce body weight and fat mass, and increase basal metabolicrate in animal models. However, studies show that CLA inducesinsulin resistance in mice and in humans, whereas Cr improvesinsulin sensitivity. Hence, we designed the present study to ex-amine the combined effect of CLA and Cr on body compositionand insulin sensitivity in a Balb/c mice (n = 10/group) model ofhigh-fat-diet–induced obesity. CLA alone lowered body weight,total body fat mass, and visceral fat mass, the last of which de-creased further with the combination of CLA and Cr. This effectwas accompanied by decreased serum leptin levels in CLA-fedand CLA + Cr–fed mice, and by higher energy expenditure (EE)and oxygen consumption (OC) in CLA + Cr–fed mice. Serumlevels of glucose, insulin, the pro-inflammatory cytokines,tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6), as wellas insulin resistance index (IRI), decreased with CLA, whereasCLA and Cr in combination had significant effects on insulinand IL-6 concentrations and IRI. In summary, CLA + Cr de-creased body weight and fat mass in high-fat-diet–fed mice,which may be associated with decreased leptin levels andhigher EE and OC.

Paper no. L9861 in Lipids 41, 437–444 (May 2006).

Obesity is one of the major avoidable risk factors for mortal-ity and morbidity, and increases the risk for type 2 diabetes,cancer, diseases associated with the cardiovascular system,and osteoarthritis in weight-bearing joints (1), leading to anenormous increase in health care costs. Decrease in energyexpenditure (EE) associated with lack of physical activity has

been identified as one of the major reasons for the spread ofthis epidemic (2–5). Obesity is usually accompanied by ab-normalities in leptin and insulin secretion and their action, to-gether with defects in lipid and carbohydrate metabolism(6,7).

CLA refers to a group of polyunsaturated FA that are posi-tional and stereoisomers of octadecadienoic acid (linoleicacid, LA, 18:2n-6, double bonds at position 9 and 12). Theyare found naturally in ruminant food products such as beef,lamb, and dairy products containing approximately 80% cis-9, trans-11 (c9t11) isomer and 10% trans-10, cis-12 (t10c12)isomer (8,9). CLA is available commercially as a mixture ofc9t11 and t10c12 isomers present in equal amounts (~35%),and has significant antiadipogenic, anticarcinogenic, antiath-erosclerotic, antiinflammatory, antidiabetic, and antihyper-tensive properties in animal models (10–18). Some recentstudies also indicate that CLA reduces body fat mass (BFM)in humans (19,20). However, evidence derived from studiesin mice and humans suggest that intake of CLA or t10c12 iso-mer alone may induce hyperglycemia, hyperinsulinemia, andinsulin resistance (IR) (16,21–25). In a recent study in humanmature adipocytes, t10c12 isomer activated nuclear factor-kappaB (NF-κB) and ERK1/2-dependent tumor necrosis fac-tor-α (TNF-α) and interleukin-6 (IL-6) production, whichsuppressed peroxisome proliferator-activated receptor-γ(PPAR-γ), leading to impaired glucose uptake. This providedsupportive evidence that the t10c12 isomer induces IR (26).

Chromium is an important trace element essential for nor-mal fat, carbohydrate, and protein metabolism (27).Chromium intake increases lean mass and decreases bodyweight and fat mass in animal studies and in humans (27,28).Moreover, it improves serum lipid levels and glucose toler-ance in rodents and in humans, and increases or potentiatesinsulin’s effects on glucose uptake and utilization (29–33) anddecreases IR (34,35).

It is well established that rodents on a high-fat diet developobesity and IR, similar to the effect of Western—style diet inhumans (36–38). Because CLA and Cr have beneficial effectson body composition, we hypothesized that intake of a lowdose of CLA and Cr, in combination, would decrease fat massin a mouse model of high-fat-diet–induced obesity. We alsohypothesized that CLA could induce hyperinsulinemia andhyperglycemia in this mouse model, which may be amelio-

*To whom correspondence should be addressed at Division of Clinical Im-munology, mail code 7874, Department of Medicine, The University ofTexas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX78229-3900. E-mail: fernandes@uthscsa.eduAbbreviations: AKP, alkaline phosphatase; BFM, total body fat mass; BLM,total body lean mass; CO, corn oil; c9t11, cis-9, trans-11; DEXA, dual en-ergy x-ray absorptiometry; EE, energy expenditure; IL, interleukin; IR, in-sulin resistance; IRI, insulin resistance index; IRK, insulin receptor kinase;IRTP, insulin receptor tyrosine phosphatase; IS, insulin sensitivity; LA,linoleic acid; NF-κB, nuclear factor-kappaB; OC, oxygen consumption;PPAR-γ, peroxisome proliferator-activated receptor-γ; SFO, safflower oil;t10C12, trans-10, cis-12; TNF, tumor necrosis factor; UCP2, uncoupling pro-tein 2; VFM, visceral fat mass.

Conjugated Linoleic Acid and Chromium Lower BodyWeight and Visceral Fat Mass in High-Fat-Diet–Fed Mice

Arunabh Bhattacharyaa,d, M. Mizanur Rahmana,d, Roger McCarterb,Marianne O’Sheac, and Gabriel Fernandesa,*

Departments of aMedicine, Division of Clinical Immunology, and bPhysiology, University of Texas Health Science Center, San Antonio, Texas, 78229, and cLoders Croklaan Lipid Nutrition,

Minneapolis, Minnesota, 55410 dThese authors contributed equally.

Copyright © 2006 by AOCS Press 437 Lipids, Vol. 41, no. 5 (2006)

rated by Cr intake. To establish this hypothesis, we conductedthe present study in Balb/c mice on a high-fat diet (20% cornoil [CO], ~35% kcal) that induced obesity. Balb/c mice sig-nificantly gain body weight and fat mass in response to high-fat diet (39). We performed a longitudinal study to measurethe interaction of CLA and Cr in their effects on body com-position utilizing dual-energy x-ray absorptiometry (DEXA)technology. We measured serum levels of glucose, insulin,leptin, adiponectin, the proinflammatory cytokines, IL-6, andTNF-α, and insulin resistance index (IRI). Because CLA in-creases EE (40,41), we also measured parameters of meta-bolic rate to determine whether any CLA + Cr–mediated de-crease in fat mass is associated with higher EE and oxygenconsumption (OC).

EXPERIMENTAL PROCEDURES

Animals and experimental diets. Six-week-old male Balb/cmice were obtained from Harlan (Indianapolis, IN). Weight—matched mice were housed in a laboratory animal care facil-ity in cages (5 mice/cage) and fed standard lab chow diet. At8 wk of age, animals were divided into different groups andprovided with semipurified AIN-93M diets containing 0.5%safflower oil (SFO) or 0.5% CLA (Clarinol powder, LodersCroklaan, Channahon, IL). Each group was further dividedinto Cr and non-Cr groups of 10 mice each, thus resulting infour treatment groups: SFO, SFO + Cr, CLA, and CLA + Cr .The diets contained 20% CO by weight. The composition ofthe semipurified diets is provided in Table 1. The composi-tion of the oil extracted from the Clarinol powder was: c9t11,36.9%; t10c12, 37.4%; total trans, 2.7%. The total CLA con-tent of the oil was 80.9%, with the main isomers accountingfor 74.4%. Like CO, SFO contains 18:2n-6 (LA) as the majorFA component. Cr was provided as chromium tricarnosinateat 22.77 g/kg diet (3.4% chromium and 89.3% carnosine, Fu-tureCeutical Carnochrome, VDF FutureCeuticals, Momence,IL). Fresh diet was prepared weekly, stored in aliquots at–20ºC, and provided daily ad libitum. Body weight was mon-itored biweekly. Animals were maintained on a 12—hr

light/12—hr dark cycle in an ambient temperature of 22–25ºCat 45% humidity. NIH guidelines (42) were strictly followed;and the Institutional Laboratory Animal Care and Use Com-mittee of the University of Texas Health Science Center atSan Antonio approved all studies.

Collection of tissues and blood serum. Blood was collectedafter overnight fasting under anesthesia by retro-orbital bleed-ing 3 d before the end of the experiment, and serum was ob-tained by centrifugation at 3000 rpm for 15 min at 4°C for thedetermination of glucose and insulin. At the termination ofthe study, mice were sacrificed by cervical dislocation, andserum was collected for all the other assays.

Measurement of fat mass and lean mass. BFM and totalbody lean mass (BLM) were measured by DEXA using aLunar PIXImus mouse bone densitometer (GE, Madison,WI), and data were analyzed with PIXImus software (43—45). Before scanning was performed, mice were anesthetizedwith an intramuscular injection of 0.1 mL/100 g body weightof mouse cocktail containing ketamine/S.A. rompun/NaCl(3:2:5, by vol). BFM and BLM were obtained for the entirebody, excluding the head. Visceral fat mass (VFM) was de-termined manually using DEXA. Scanning was performedfirst at baseline and again at the end of 14 wk of the study diet.

Serum metabolites. Insulin was analyzed using a rat/mouseinsulin ELISA kit from Linco Research (St Charles, MO).Glucose was analyzed spectrophotometrically using SigmaDiagnostics glucose (Trinder) reagent (Sigma Diagnostics, StLouis, MO). Adiponectin was assayed using mouseadiponectin Quantikine immunoassay kit from R&D Systems(Minneapolis, MN). Leptin was assayed using an activemurine leptin kit from Diagnostic Systems Laboratories(Webster, TX).

IRI. IRI was calculated by the following formula: [fastingserum insulin (ng/mL) × fasting serum glucose (mM)]/22.5.A high IRI denotes low insulin sensitivity (IS) (46).

Serum proinflammatory cytokines. TNF-α and IL-6 weremeasured by using BD OptEIA™ ELISA kits from BD Bio-sciences Pharmingen (San Diego, CA) (43). Sensitivity of theassays was approximately 1 pg/mL. In brief, each well of flat-bottom 96-well microtiter plates was coated with 100 µl ofpurified anti-TNF-α and anti-IL-6 antibodies (4 µg/mL inbinding solution) overnight at 4°C. The plates were rinsedfour times with washing buffer, and diluted serum was added,followed by incubation for 2 h at room temperature. Theplates were washed four times with washing buffer, followedby the addition of biotinylated anticytokine antibodies. Theplates were incubated in room temperature for 1 h and thenwashed four times with washing buffer. Streptavidin-alkalinephosphatase conjugate was added, and the plates were incu-bated for 30 min at room temperature. The plates were againwashed four times with washing buffer, and the chromogensubstrate was added. The plates were then incubated at roomtemperature to achieve the desired maximum absorbance andwere read at 410 nM in an ELISA reader (Dynex Technolo-gies, Worthington, West Sussex, United Kingdom).

Metabolic rate. Metabolic rate was measured indirectly by

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TABLE 1Composition of Experimental Diets

Ingredients (g/kg) SFO CLA SFO + Cr CLA + Cr

Casein 248 248 248 248AIN-93 mineral mix 64 64 64 64AIN-93 vitamin mix 17 17 17 17Cellulose 55 55 55 55Anhydrous dextrose 405.5 403.74 382.73 380.97Choline bitartrate 2.5 2.5 2.5 2.5DL-Methionine 3.0 3.0 3.0 3.0Clarinol powdera 0 6.76 0 6.76Safflower oil 5.0 0 5.0 0Chromium cereloseb 0 0 22.77 22.77Corn oil 200 200 200 200

Total 1000 1000 1000 1000aContains 74% CLA.bCarnoChrome (chromium tricarnosinate), 3.14% (wt/wt); chromium mixed3 mg/10 g in cerelose.

drawing air through sealed rodent cages and monitoring thepartial pressures of oxygen and carbon dioxide of the air en-tering and leaving the cage, as described in several earlierstudies (47). In brief, gas composition was measured using azirconia cell O2 detector and an infrared CO2 analyzer (Ap-plied Electrochemistry models S-3A and CD-3A, respec-tively; Ametek, Paoli, PA), with flow regulated by a massflow controller (Linde model FM4570; Ontario, Canada). Gaspressures, airflow, cage temperature, ambient air temperature,pressure, and humidity were digitized, recorded every 10 s,and averaged on an hourly basis. Each mouse was monitoredbetween 0800 and 1200 h for 3 h. Mice were acclimated tothe flowthrough system for 24 h before measurement of meta-bolic rate. During the time of measurement, air was also sam-pled from an empty cage situated adjacent to the experimen-tal cage, as a control. The rate of O2 consumption was calcu-lated using the equations of Consolazio et al. (48). Themetabolic rate was then calculated in kJ/min using these val-ues and an energy equivalent of 1 L of oxygen per 4.76 kJ.This assumes that the fuel combusted in vivo is the same asthat of the food ingested; only small errors are introduced ifthe fuel mix metabolized at a given time deviates from thisvalue.

Statistical analysis. Results are expressed as means ±SEM. Data were analyzed statistically by two-way ANOVAor repeated-measures ANOVA using Graphpad Prism 4 soft-ware. The Newman-Keuls multiple comparison test was usedto test the differences among groups. Differences were con-sidered significant at P < 0.05.

RESULTS

Body composition and liver weight. Body weight initially in-creased in all the dietary groups until 6 wk of age. Thereafter,body weight increased maximally in SFO-fed mice, whereasbody weight decreased in CLA + Cr–fed mice (Fig. 1). Bodyweight was significantly lowered in CLA-fed mice, comparedwith SFO-fed mice (P < 0.001). Cr decreased body weight inboth SFO-fed and CLA-fed mice but more significantly inCLA + Cr–fed mice. Body weight in SFO + Cr–fed mice wascomparable to CLA-fed mice. CLA-fed mice showed lowerfat mass that decreased further in CLA + Cr–fed mice (P <0.05) compared with SFO-fed mice. VFM is the sum of theweights of the mesenteric, epididymal, and retroperitoneal fatdepots. VFM was significantly lowered in CLA-fed micecompared with SFO-fed mice, and decreased further in CLA+ Cr–fed mice (P < 0.05). There was no difference in liverweights between the different dietary groups (Table 2). Leanmass was higher in CLA-fed mice compared with SFO +Cr–fed and CLA + Cr–fed mice but not SFO-fed mice (Table2).

Serum metabolites. Leptin was lowered in CLA-fed andCLA + Cr–fed mice compared with SFO-fed and SFO +Cr–fed mice but there was no effect of Cr supplementation (P< 0.001). Serum adiponectin levels were unaltered by CLAor Cr. Glucose concentrations was lowered in CLA-fed mice

compared with SFO-fed and SFO + Cr–fed mice (P < 0.05).However, glucose level in the CLA + Cr group was compara-ble to that in all the other dietary groups. Analysis by two-way ANOVA for main effects revealed that the only statisti-cally significant effect of CLA was on glucose level. Insulinlevels decreased in SFO + Cr–fed, CLA-fed, and CLA +Cr–fed mice compared with SFO-fed mice. Analyzing thedata by two-way ANOVA for main effects showed that CLAand the combination of CLA and Cr had significant effects oninsulin levels. Insulin sensitivity was estimated by several in-dices, including the homeostasis model assessment, l/insulin,and glucose/insulin. A higher IRI reflects low IS; IRI waslowered in all the other dietary groups compared with SFO-fed mice. However, the decrease was much higher in CLA-fed and CLA + Cr–fed mice. Analyzing the data by two-wayANOVA for main effects showed significant effects of CLA,Cr, and their interaction on IRI (Table 3).

Serum proinflammatory cytokines. Serum TNF-α levelswere lowered in CLA-fed and CLA + Cr–fed mice comparedwith SFO-fed and SFO + Cr–fed mice (P < 0.05), but therewas no additional effect of Cr. Two-way ANOVA for maineffects revealed that the only statistically significant effect ofCLA was on TNF-α, whereas interaction of CLA and Cr wasclose to being statistically significant. Serum IL-6 levels weredecreased in SFO + Cr–fed, CLA-fed, and CLA + Cr–fedmice compared with SFO-fed mice. Cr lowered IL-6 in SFO-fed mice but there was no additional effect of Cr in CLA-fedmice. Analyzing the data by two-way ANOVA for main ef-fects found significant effects of CLA and interaction of CLAand Cr on IL-6 activity (Table 3).

OC and EE. OC and EE did not differ between SFO-fed

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FIG. 1. Body weight curves for safflower oil (SFO) control, conjugatedlinoleic acid (CLA)–treated, SFO and chromium (Cr) (SFO + Cr)–treated,and CLA and Cr (CLA + Cr)–treated mice (n = 10/group). Day 0 was thefirst day mice were placed on different diets. Both CLA and Cr signifi-cantly suppressed body weight (CLA: P < 0.0001; Cr: P < 0.05).

and CLA-fed mice. However, both OC and EE were higher(P < 0.05) in CLA + Cr–fed mice than in SFO-fed mice. An-alyzing the EE data by two-way ANOVA for main effects re-vealed statistically significant effects of CLA alone and Cralone but not in combination (Table 4).

DISCUSSION

High-fat—diet–induced obesity is often associated with IR,which is a major risk factor for diabetes (49,50). Visceral obe-sity, in particular, has a strong correlation with IR and onsetof glucose intolerance (51). Rats and mice fed a high-fat diethave increased VFM, whole body and muscle IR, and hyper-insulinemia within 4 wk, whereas continuous feeding of ahigh-fat diet for 8–12 wk develops severe visceral obesity, di-

abetes or impaired glucose tolerance, and plasma lipid andlipoprotein abnormalities (38). In the present study, CLA de-creased body weight, BFM, and VFM, the last of which de-creased further with the combination of CLA and Cr. Previ-ous studies in animals fed CLA or the t10c12 isomer haveshown decreased body weight and fat mass compared withcontrol animals (40,52—54). Cr alone decreased body weightand fat mass in our study in accordance with previous studies(27). This is the first observation to show that Cr has benefi-cial effects on body composition in high-fat-diet–fed mice.

The antiadiposity effect of CLA has been ascribed, in part,to its ability to alter energy balance. Some studies in mice haveshown that decreased energy intake may be insufficient to ac-count for CLA—mediated dramatic loss of fat mass, implyingthat other mechanisms may be involved (40,41,52). Increased

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TABLE 3Serum Parameters in Male Balb/c Mice Fed Control (SFO) or CLA Diets With or Without Chromium (Cr) Supplementation for 14 wka

P Valueb

SFO CLA SFO + Cr CLA + Cr CLA Cr CLA × Cr

Glucose mg/dL 162.70 ± 6.15a 138.10 ± 4.55b 169.30 ± 5.66a 154.80 ± 6.58ab <0.01 0.06 0.39mM 9.02 ± 0.34a 7.66 ± 0.25b 9.39 ± 0.31a 8.59 ± 0.36ab <0.01 0.06 0.39Insulin (ng/mL) 20.10 ± 2.02a 7.60 ± 0.31b 11.64 ± 0.74b 11.04 ± 1.49b <0.001 0.08 <0.001Insulin Resistance Indexc 7.96 ± 0.58a 2.59 ± 0.17b 5.11 ± 0.07c 3.66 ± 0.49b <0.0001 0.04 <0.001

(ng/mL × mM)Leptin (ng/mL) 2.42 ± 0.06a 1.61 ± 0.07b 2.45 ± 0.10a 1.91 ± 0.16b <0.0001 0.13 0.23Adiponectin (µg/mL) 3.53 ± 0.32 3.88 ± 0.11 4.03 ± 0.14 3.90 ± 0.28 0.61 0.28 0.32TNF-α (pg/mL) 252.8 ± 18.21a 108.5 ± 2.83b 199.23 ± 1.50a 131.0 ± 16.15b <0.001 0.46 0.08IL-6 (pg/mL) 106.00 ± 10.97a 64.50 ± 1.46b 80.63 ± 1.88b 71.00 ± 3.92b <0.01 0.16 0.02

aValues are means ± SEM, n = 5. Means in the same row with superscripts without a common letter differ by Newman–Keuls multiple comparison test, P <0.05. bFrom two-way ANOVA.cFasting glucose (mM) × fasting serum insulin (ng/mL)/22.5.

TABLE 2Body Composition and Liver Weights in Male Balb/c Mice Fed Control (SFO) or CLA Diets With or Without Chromium (Cr) Supplementationfor 14 wka

P Valueb

SFO CLA SFO + Cr CLA + Cr CLA Cr CLA × Cr

Body weight, gBaseline 25.64 ± 0.80 24.67 ± 0.73 27.84 ± 0.87 27.08 ± 0.99Final 34.67 ± 1.20a 30.19 ± 0.58bc 32.66 ± 0.88ac 28.35 ± 0.59b <0.0001 0.04 0.93Increase in body weight 9.03 ± 0.93a 5.54 ± 0.46b 4.82 ± 0.77b 1.25 ± 0.49c

Total body fat mass, gBaseline 3.06 ± 0.15a 2.47 ± 0.07b 3.57 ± 0.17c 3.69 ± 0.22c

Final 8.10 ± 0.75a 3.74 ± 0.33b 5.91 ± 0.74c 3.13 ± 0.24b <0.0001 0.03 0.20Increase in fat mass 5.02 ± 0.65a 1.24 ± 0.33b 2.34 ± 0.85b –0.45 ± 0.24c

Total body lean mass, gBaseline 16.38 ± 0.19a 16.23 ± 0.22a 17.63 ± 0.32b 17.40 ± 0.33b

Final 19.39 ± 0.55ab 20.25 ± 0.38a 20.48 ± 0.30a 18.71 ± 0.34b 0.28 0.60 <0.01Increase in lean mass 3.01 ± 0.53ab 4.08 ± 0.31b 2.87 ± 0.32a 1.04 ± 0.42c

Visceral fat mass, gBaseline 0.77 ± 0.07 0.50 ± 0.04 0.89 ± 0.06 1.00 ± 0.08Final 2.65 ± 0.16a 0.81 ± 0.17b 1.72 ± 0.16c 0.66 ± 0.18b <0.0001 0.04 0.15Increase in visceral fat mass, g 1.87 ± 0.13a 0.30 ± 0.05b 0.83 ± 0.06c –0.32 ± 0.08d

Liver weight, g 1.37 ± 0.08 1.44 ± 0.07 1.41 ± 0.04 1.47 ± 0.05 0.13 0.22 0.24aValues are means ± SEM, n = 10. Means in a row with superscripts without a common letter differ by Newman–Keuls multiple comparison test, P < 0.05. bFrom two-way ANOVA with repeated measures at 14 wk.

EE has increasingly been proposed to be one of the mecha-nisms for the antiadiposity effects of CLA and particularly withthe use of t10c12 isomer (40,41). Research suggests that un-coupling protein 2 (UCP2) expressed in white and brown adi-pose tissues may play a significant role in regulation of EE byCLA. UCP2 mRNA expression was shown to be augmentedby CLA and t10c12 isomer in white adipose tissues from miceand rats, and in cultured adipocytes (21,22,52). We observedhigher EE and OC in CLA + Cr–fed mice compared with con-trol diet–fed mice, suggesting that CLA + Cr–mediated loss offat mass may be associated, in part, with increased EE and OC.

Leptin, a product of the obese gene, is secreted primarilyby adipocytes and plays an important role in regulating en-ergy balance. Circulating leptin is highly correlated with gen-eral adiposity in obese rodents and individuals exhibitinghigher plasma leptin levels (55,56). Decreased leptin levelsin CLA-fed mice were correlated with decrease in fat mass inthe present study, suggesting that this may be one of themechanisms involved in the observation. Because Cr did notlower circulating leptin levels in our study, Cr—induced lossof fat mass may be independent of leptin concentration. CLAand Cr have been reported to decrease serum leptin levels inanimal studies (57,58). We have previously shown that CLAdecreases leptin mRNA expression in peritoneal fat tissues,and CLA also decreases circulating leptin levels in high-fat-diet–fed mice (44).

In the present study, decreased glucose, insulin, and IRIwere observed in CLA-fed mice, indicating a beneficial ef-fect on IS. This is indeed an interesting finding consideringthat CLA and t10c12 isomer alone induced hyperinsulinemia,hyperglycemia, and IR in most of the studies in mice(21,22,59) and in humans (23–25). Studies suggest thatPPAR-γ may be one of the key modulators of IR by CLA(more predominantly by the t10c12 isomer). PPAR-γ is down-regulated in both adipose tissues from CLA-fed mice and cul-tured adipocytes. Downregulation of PPAR-γ contributes toloss of adiposity and decreased IS (21,60). Recently, Chunget al. found that t10c12 isomer decreases PPAR-γ expressionin human adipocytes through NF-κB activation (26). Thisstudy provides evidence that inhibition of PPAR-γ in humanadipocytes by CLA and/or t10c12 isomer may be a key mech-anism involved in IR in humans. Interestingly, studies in nor-mal and diabetic rats indicate beneficial effects of CLA andt10c12 isomer on glucose and insulin levels, glucose toler-

ance, and IS (52,61,62), which could be related to activationof PPAR-γ in these studies (61,63). Thus, the effect of CLAon IR seems to be dependent partly on species, metabolicstate of the animal, and response of PPAR-γ to CLA, morespecifically the t10c12 isomer. Wargent et al. recently demon-strated increased glucose and insulin levels, and exacerbatedglucose tolerance, in genetically obese (lepob/lepob) mice fedCLA or t10c12 isomer–enriched CLA for 2 wk. However,long-term CLA intervention (10 wk) had beneficial effects onglucose, insulin, and IRI levels (64). This study and long-termstudies in humans suggest that the detrimental effect of CLAon IR may be a transient effect seen in short-term studies;long-term studies did not show any adverse effects on glucoseand insulin levels (19,20). However, these findings need to beconfirmed with more long-term studies in rodents and humansusing purified isomers. Cr alone significantly decreased in-sulin level and IRI in our study, in accordance with previousstudies (34,57). Our results further suggest that CLA + Crmay have additive beneficial effects on insulin level and IRIin high-fat-diet–fed mice. Although the results from the pre-sent study are encouraging and suggest a beneficial effect ofCLA + Cr on IS, more mechanistic studies in animal modelsneed to be pursued to establish the efficacy of CLA + Cr sup-plementation on IR and body composition. Cr has beenshown to activate insulin receptor kinase (IRK) but to down-regulate insulin receptor tyrosine phosphatase (IRTP) (33).The activation of IRK and inhibition of IRTP may lead to in-creased phosphorylation of the insulin receptor and improvedIS. Because PPAR-γ is an important target for CLA, studyingthe effect of Cr on PPAR-γ in adipocytes derived from rodentsand humans may be of interest to establish the beneficial ef-fect of Cr on IR in mice fed CLA.

Adiponectin is secreted by fat cells and enhances insulinaction by suppressing hepatic glucose production and glucoseuptake by skeletal muscles (65). In human studies,adiponectin levels are negatively correlated with fat mass(65,66). However, in a recent study in PPAR-γ adipose knock-out mice, improved IS was observed along with decreasedadipose tissue expression and lower plasma levels ofadiponectin, suggesting that adiponectin produced by fat maynot completely account for the improved IS (67). Moreover,Baratta et al. reported that the plasma adiponectin level wasindependent of fat mass in nondiabetic and diabetic patients(68). CLA and Cr alone or in combination had no effect on

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TABLE 4Oxygen Consumption and Energy Expenditure in Male Balb/c Mice Fed Control (SFO) or CLA Diets With or Without Chromium (Cr)Supplementation for 14 wka

P Valueb

SFO CLA SFO + Cr CLA + Cr CLA Cr CLA × Cr

Energy expenditure 11.29 ± 0.45a 13.11 ± 0.44ab 13.29 ± 0.54ab 14.15 ± 0.90b 0.04 0.03 0.44(kcal/kg body weight)

Oxygen consumption 3.01 ± 0.12a 3.50 ± 0.12ab 3.54 ± 0.14ab 3.77 ± 0.24b 0.16 0.21 0.89(L O2/kg body weight)

aValues are means ± SEM, n = 5. Means in the same row with superscripts without a common letter differ by Newman–Keuls multiple comparison test, P <0.05. bFrom two-way ANOVA.

serum adiponectin levels in the present study. Our previousstudy on the effects of CLA and exercise interaction in high-fat-diet–fed mice also did not show any effect on serumadiponectin levels or adiponectin mRNA expression in peri-toneal fat pads derived from these mice (44). Adiponectin hasbeen shown to be increased in CLA—fed diabetic rats but de-creased in mice, which was associated with decreased IR(69—71). Improved IS without enhanced serum adiponectinlevels in our study suggest that beneficial effects of CLA maybe independent of adiponectin concentrations in this mousemodel.

CLA alone and in interaction with Cr had beneficial effectson serum TNF-α and IL-6 levels in the present study. Our pre-vious study in peritoneal fat derived from high-fat-diet–fedmice clearly indicated a statistically significant effect of CLAon TNF-α mRNA expression, which suggests that inhibitionmay occur at the adipose tissue level (44). CLA has been pre-viously shown to decrease TNF-α and IL-6 in serum andmacrophages derived from rats and mice (58,63,70,72),whereas other studies have reported either no effect or signif-icant elevation of these cytokines in cultured cells from miceand humans (73,74). Cr has been reported to inhibit endo-toxin—mediated inflammatory cytokine production in swine(75) and phytohemagglutinin—stimulated human peripheralblood mononuclear cells (76). The present data suggests thatCLA and Cr may have additive effects in decreasing TNF-αand IL-6 in high-fat-diet–fed mice, which, in part, could ex-plain improved IS in these mice.

We did not observe any enlarged livers in CLA—fed mice,although there was a dramatic decrease in fat mass. Previousstudies show that CLA and t10c12 isomer increase liverweight in mice due to increased fat deposition (21,59). How-ever, when CLA was supplemented with a high-fat diet, themice did not show higher liver weight compared with controlmice (77). A similar phenomenon may have influenced liverweights in this study.

In conclusion, the present study suggests that the combi-nation of CLA and Cr lowers body weight and VFM, and mayhave a beneficial effect on IS in high-fat—diet–fed mice.More studies in different strains of mice and other species arewarranted before the combination of CLA and Cr may be rec-ommended to be beneficial in human health.

ACKNOWLEDGMENTS

This study was supported by National Institutes of Health grants(AG023648 and AG027562) and a grant from Loders Croklaan, Inc.

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[Received September 6, 2005; accepted May 29, 2006]

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