Int. J. Mol. Sci. 2013, 14, 6848-6862; doi:10.3390/ijms14046848 Article OPEN ACCESS Interntionl Journl of Moleculr Sciences ISSN 1422-0067 www.mdpi.com/journl/ijms Effects of Dietry Conjugted Linoleic Acid nd Biopolymer Encpsultion on Lipid Metolism in Mice Sun Jin Hur, Doo Hwn Kim, Se Chul Chun nd Si Kyung Lee * Deprtment of Bioresources nd Food Science, Konkuk University, 120 Neungdong-ro, Gwngjin-gu, Seoul 143-701, Kore; E-Mils: sjhur@konkuk.c.kr (S.J.H.); kimdh@konkuk.c.kr (D.H.K.); scchun@konkuk.c.kr (S.C.C.) * Author to whom correspondence should e ddressed; E-Mil: lesikyung@konkuk.c.kr; Tel.: +82-2-450-3759; Fx: +82-2-450-3726. Received: 17 Jnury 2013; in revised form: 11 Mrch 2013 / Accepted: 19 Mrch 2013 / Pulished: 26 Mrch 2013 Astrct: Forty mice were rndomly divided into four groups on the sis of the diet to e fed s follows: 5% (low) ft diet (T1: LF); 20% (high) ft diet (T2: HF); 20% ft contining 1% conjugted linoleic cid (CLA) (T3: HFC); nd 20% ft contining 1% CLA with 0.5% iopolymers (T4: HFCB). The high-ft with CLA diet groups (HFC nd HFCB) nd the low-ft diet group (LF) tended to hve lower ody weights nd totl dipose tissue weights thn those of the high-ft diet group (HF). Serum leptin nd triglyceride were significntly lower in the high ft with CLA-fed groups (HFC nd HFCB) nd the low-ft diet group (LF) thn those in the high-ft diet group (HF). It is noteworthy tht the high-ft with CLA nd iopolymers group (HFCB) showed the lowest serum triglyceride nd cholesterol concentrtions. In the high-ft-fed group (HF), voluntry trvel distnce s mesure of physicl ctivity decresed fter three weeks of feeding. However, the CLA-fed groups showed incresed physicl ctivity. The groups fed high-ft diets supplemented with CLA lone nd with CLA nd iopolymers hd higher viscosity of smll intestinl contents thn tht in the low- nd high-ft dietry groups. Keywords: conjugted linoleic cid; iopolymers encpsultion; energy expenditure; dipose tissue; lipid metolism
Int. J. Mol. Sci. 2013, 14 6849 1. Introduction Oesity nd eing overweight cn occur when energy intke is higher thn energy expenditure. It hs long een proposed tht incresed energy expenditure due to physicl ctivity my e the most importnt fctor in ody weight control. Pesctello nd VnHeest [1] reported tht increse in oesity is due to reltively greter decrese in physicl ctivity rther thn due to n increse in energy intke. Westerterp-Plnteng [2] reported tht ft intke ws relted to reltively lower stiety nd tht the ft content of food ppers to ffect mel frequency. In generl, ft hs higher cloric density thn proteins nd crohydrtes, nd its contriution to the pltility of foods promotes ingestion of clories [3]. Hill et l. [4] reported tht consumption of high-ft diet increses the totl energy intke nd tht excess dietry ft is stored with greter efficiency thn excess dietry crohydrte or protein. Horton et l. [5] lso reported tht greter proportion of excess energy is stored during ft overfeeding (pproximtely 90% 95%) thn tht with n equivlent mount of crohydrte overfeeding (pproximtely 75% 85%). Thus, n increse in excessive energy intke from ft cn reduce physicl ctivity, nd this decline of physicl ctivity cuses oesity [1]. Thus, it cn e stted tht energy sources ply n importnt role in weight gin nd oesity. In the mentime, Prk et l. [6] first reported the influence of conjugted linoleic cid (CLA) on ody composition in study in which mice fed diet with 0.5% CLA hd decresed ody ft mss nd incresed len ody mss. Numerous studies using vrious models, including humn models, hve reported the effect of CLA on ody composition or ft reduction. Our previous study found tht CLA supplementtion decresed overll ody weight nd totl ft nd tht the effect of dietry CLA on dipose tissue reduction ws greter in n oese mouse model thn tht in wild-type mouse model [7]. Despite the lrge numer of studies showing tht CLA increses energy expenditure, s shown y incresed oxygen consumption [8 10], how CLA influences ody weight or ody ft in terms of different cloric nd ft contents in niml models is poorly understood. Numerous studies hve demonstrted tht the ility of vrious iopolymers cting s dietry fiers, such s chitosn, pectin, gur gum, xnthn gum or modified strch, cn lower lood cholesterol concentrtions nd reduce lipid sorption. Dietry fiers could decrese serum cholesterol concentrtions y reducing the mount of sored cholesterol. Furthermore, dietry fiers my interfere with the formtion of lipid droplets, or they my interfere with the ility of lipse to ccess the lipids contined within the lipid droplets. In n erly study, Liron [11] reported tht dietry fiers cn lter the rekup nd colescence of lipid droplets in the stomch nd smll intestine, therey ltering the surfce re of emulsified lipid exposed to digestive enzymes. In the sme study, the uthors reported tht vrious dietry fier sources cn ind to ile cids, s well s to mixed micelle components; thus, they explined the prticle disruption of the micelliztion process, which leds to reduced micellr soluiliztion of lipids. For instnce, ctionic chitosn cn ind to the surfces of nionic lipid droplets stilized y ile slt or phospholipids nd reduce lipse ctivity y preventing lipse from coming into contct with emulsified lipid sustrtes [12]. However, how iopolymer encpsultion influences lipid metolism in CLA dietry model remins to e elucidted. In ddition, there is little informtion on how dietry CLA influences lipid metolism in models fed the sme mount of clories. Thus, the purpose of this study ws to determine the effects of CLA nd iopolymers on lipid metolism in mice fed high- nd low-ft diets.
Int. J. Mol. Sci. 2013, 14 6850 2. Results 2.1. Food Intke nd Body Weight There were no significnt differences in food intke mong the dietry groups (Figures 1 nd 2). After three weeks of feeding, the high-ft with CLA diet groups (HFC nd HFCB) nd the low-ft diet group (LF) tended to hve lower ody weights thn tht of the control group (HF). This result indictes tht low- nd high-ft diets with CLA cn suppress ody weight gin; this is despite the fct tht totl clorie levels were the sme for oth the low- (5%) nd high-ft (5%) diet groups. Figure 1. Effect of inding of conjugted linoleic cid with iopolymers on food intkes in mice fed high- nd low-ft diets. Significnce ws set t p < 0.05. 40 Food intke (g/mouse/week) 35 30 25 20 15 T1 T2 T3 T4 0 1 2 3 4 5 6 Feeding periods (weeks) Figure 2. Effect of inding of conjugted linoleic cid with iopolymers on ody weight in mice fed high- nd low-ft diets. Significnce ws set t p < 0.05. 50 Body weight (g) 45 40 35 30 c c 25 T1 T2 T3 T4 20 0 1 2 3 4 5 6 Feeding periods (weeks) 2.2. Adipose Tissue Weigh Totl dipose tissue weight ws clculted s the sum of perimetril, mesenteric nd retroperitonel dipose tissue weights (Figure 3). The group fed high-ft CLA diet (HFC) nd the group fed
Int. J. Mol. Sci. 2013, 14 6851 high-ft CLA nd iopolymer diet (HFCB) hd significntly lower totl dipose tissue weights thn their control groups (LF nd HF). Figure 3. Effect of inding of conjugted linoleic cid with iopolymers on dipose tissue weights in mice fed high- nd low-ft diets. Significnce ws set t p < 0.05. 7 6 Adipose tissue weight (g) 5 4 3 2 T1 T2 T3 T4 c c 1 0 Perimetril Mesenteric Retroperitonel Totl dipose tissue 2.3. Blood Chrcteristics Tle 1 shows the effect of dietry CLA with iopolymers on lood chrcteristics. Serum leptin concentrtions were significntly lower in the groups fed high-ft CLA diets (HFC nd HFCB) nd in the low-ft diet group (LF) thn those in the high-ft diet group (HF); conversely, diponectin ws significntly higher in the CLA diet groups (HFC nd HFCB) thn tht in the other dietry groups. Serum triglyceride nd cholesterol concentrtions were significntly lower in the high-ft diet with CLA groups (HFC nd HFCB) thn those in the control group (HF). It is noteworthy tht the group fed the high ft with CLA nd iopolymers diet (HFCB) hd the lowest serum triglyceride nd cholesterol concentrtions mong ll the dietry groups. Glucose concentrtions were significntly higher in the low-ft diet group (LF) thn those in the high-ft diet groups (HF, HFC nd HFCB). Tle 1. Effect of inding of conjugted linoleic cid with iopolymers on lood chrcteristics in mice fed high- nd low-ft diets. Test items Tretments Leptin Adiponectin Triglyceride Cholesterol Glucose (ng/ml) (μg/dl) (mg/dl) (mg/dl) (mg/dl) T 1 72.12,c ± 4.33 11.69 ± 4.80 112.24 c ± 11.29 150.56 ± 11.87 57.24 ± 2.31 T 2 118.36 ± 3.37 16.41 ± 6.09 179.17 ± 10.23 176.64 ± 12.36 50.50 ± 1.23 T 3 62.23 c ± 6.42 30.36 ± 4.23 151.46 ± 9.45 168.38 ± 10.19 48.36 ± 4.97 T 4 77.75 ± 7.37 25.22 ± 5.37 120.27 c ± 13.78 122.71 c ± 9.97 51.78 ± 3.45 d Mens with different superscripts in the sme column re significntly different (p < 0.05).
Int. J. Mol. Sci. 2013, 14 6852 2.4. Voluntry Trvel Distnce Figure 4 shows the effect of diet supplemented with CLA nd iopolymers on voluntry trvel distnce. Voluntry trvel distnce s mesure of physicl ctivity decresed fter three weeks of feeding in the high-ft fed group (HF); however, the CLA-fed groups (HFC nd HFCB) displyed slight increse in physicl ctivity, lthough the ft content of these groups ws higher thn tht of HF. Incresing physicl ctivity is n importnt strtegy for preventing oth weight gin nd weight regin [13]. However, there is little scientific informtion s to how much physicl ctivity is required to prevent weight gin in mice. In the present study, we found tht low-ft (5%) diet increses the voluntry trvel distnce compred to high-ft (20%) diet. Mice fed high-ft diet supplemented with CLA (HFC nd HFCB) showed slightly higher levels of voluntry trvel distnce thn mice in the other dietry groups. Figure 4. Effect of inding of conjugted linoleic cid with iopolymers on voluntry trvel distnce in mice fed high- nd low-ft diets. Significnce ws set t p < 0.05. 700 600 T1 T2 T3 T4 Trvel distnce (m/dy) 500 400 300 200 100 c c 0 0 1 2 3 4 5 6 Feeding periods (weeks) 2.5. Viscosity of Smll Intestine Contents Figure 5 shows the effect of dietry CLA with iopolymers on the viscosity of smll intestine contents. The viscosity of smll intestinl contents ws higher in the 20% ft diet groups (HF, HFC nd HFCB) thn tht in the 5% ft diet group (LF). The dietry CLA nd iopolymers groups (HFC nd HFCB) showed higher viscosity of smll intestinl contents thn tht of the other dietry groups (LF nd HF).
Int. J. Mol. Sci. 2013, 14 6853 Figure 5. Effect of inding of conjugted linoleic cid with iopolymers on the viscosity of smll intestine contents in mice fed high- nd low-ft diets. Significnce ws set t p < 0.05. T1 T2 T3 T4 3. Discussion Incresed food intke nd decresed energy expenditure my increse ody weight. Westerterp-Plnteng [2] reported tht the ft content of food ppers to ffect mel frequency. However, in the current study, we found no significnt difference in food intke mong the dietry groups. This my e ecuse the totl dietry clories were the sme for the 5% nd 20% ft diet groups. Sneddon et l. [14] reported tht the verge dily food intkes did not ecome significntly different until weeks three of experiment, indicting tht pltility of the diets ws not mjor issue in CLA fed rts. Thus, chnges in lipid metolism resulting from dietry CLA nd iopolymer encpsultion my not e the result of different food intke mounts in the present study. Moreover, CLA lone my hve een responsile for the reduction of dipose tissue mss or lipid metolism oserved in this study. Westerterp-Plnteng [2] reported tht energy intke from ft hs greter effect on ody weight thn energy from non-ft sources. Hill et l. [4] reported tht consumption of high-ft diet increses the totl energy intke nd tht excess dietry ft is stored with greter efficiency thn similr dietry crohydrte or protein excess. In this study, increses in ody weight my hve een more influenced y ft sources thn crohydrte sources under the sme clorie conditions. There re severl possile mechnisms for this reduced ody weight in response to dietry CLA. We ssumed tht the mjor ody weight reducing effect in mice ws the result of decresed dipose tissue weight. This my e closely relted with incresed energy expenditure cused y incresed voluntry ctivity, such s trvel distnce. Prk et l. [15] reported tht dietry CLA cn
Int. J. Mol. Sci. 2013, 14 6854 reduce dipose cell mss nd/or cell numers nd cn cuse inhiition of steroyl-coa desturse, s well s incresed ftty cid β-oxidtion in skeletl muscle. This incresed ftty cid β-oxidtion in skeletl muscle my e relted to incresed energy expenditure cused y dietry CLA. There re mny possile mechnisms tht my explin reduced ody ft in response to CLA tretments. In n erlier study, Prk et l. [6] reported tht CLA increses ft oxidtion nd this is lso supported y incresed crnitine plmitoyltrnsferse ctivity in the ft pds, muscles nd liver of CLA-fed mice. Those uthors lso reported triglyceride uptke nd storge in dipose depots, s CLA hs een shown to inhiit lipoprotein lipse ctivity in 3T3-L1 dipocytes [6]. Another possile mechnism for the reduced ody ft seen fter dietry CLA intke my e energy expenditure. Our findings in the present study gree with their study. In the present study, the levels of voluntry trvel distnce were significntly greter in CLA-fed mice thn those in control group mice, regrdless of the mount of ft. This increse in voluntry trvel distnce s mesure of physicl ctivity cused y dietry CLA resulted in incresed energy expenditure nd dipose tissue depletion. This my e one of the min mechnisms involved in the ft reduction effect of CLA. However, we sw no significnt difference in dipose tissue weight etween the group fed with high-ft CLA diet nd the group fed with high-ft CLA nd iopolymer diet. This finding indictes tht dipose tissue depletion my originte from CLA itself nd not from iopolymers. Leptin is protein hormone with importnt effects on the regultion of ody weight nd lipid metolism. It is secreted y dipocytes in proportion to the mount of lipid stored nd my function s signl of ody energy stores to the rin [16]. Severl studies hve reported the effects of dietry CLA on leptin concentrtions. Bhttchr et l. [17] suggested tht CLA nd exercise in comintion my reduce serum leptin nd peritonel ft mrna expression, which my explin, in prt, the lowest chnge in ft mss in CLA/exercise mice. They lso suggested tht CLA my hve role in meliorting the dverse effects of exercise on the immune response [17]. Sneddon et l. [14] reported tht incresed leptin trnsport into the rin should not result in the gene chnges oserved in the hypothlmus or in the incresed food intke exhiited. In ddition, circulting diponectin, which cts centrlly to increse food intke, is reduced y pproximtely 40% with CLA plus n-3 long chin polyunsturted ftty cid supplementtion [14]. In our previous study [7], we found tht diet supplemented with CLA reduced serum leptin concentrtions in n oese murine model. The findings of these previous studies suggest tht depletion in ft mss my e due to the ssocition of CLA with reduced serum leptin concentrtions. In this study, leptin concentrtions were decresed y dietry CLA. This my result from the effect of CLA itself rther thn its metolites [15]. Thus, one of the min resons for serum leptin depletion through dietry CLA supplementtion in this study my e the lower dipose tissue content. Brown et l. [18] reported tht in humn dipose tissue, the trns-10, cis-12 isomer of CLA decreses tricylglyceride concentrtions. Bumgrd et l. [19] reported tht dietry CLA inhiits trnscription of enzymes involved in de novo ftty cid synthesis, desturtion of ftty cids nd triglyceride synthesis. Our findings in the present study gree with these results. In this study, serum glucose concentrtions were not significntly different mong the groups fed high-ft diet (HF, HFC nd HFCB). Conversely, Roche et l. [20] reported tht trns-10, cis-12 supplementtion in mice ws ssocited with incresed serum glucose nd insulin concentrtions, wheres the cis-9, trns-11 supplementtion group showed no weight loss, ut hd lower concentrtions of triglyceride nd free
Int. J. Mol. Sci. 2013, 14 6855 ftty cids. Lee et l. [21] reported tht the effect of CLA on lood glucose concentrtion ws inconsistent. Thus, we ssume tht the effect of CLA on glucose my depend on vrious fctors, such s mixture of CLA isomers, ft content in the diet, niml model, genotype nd feeding period. In this study, serum triglyceride nd cholesterol concentrtions decresed in mice fed high-ft diet supplemented y CLA nd iopolymers. In previous studies, Riserus et l. [22] found tht HDL-cholesterol concentrtions significntly decresed when the trn-10, cis-12 isomer ws dministered, ut not when mixture of the trns-10, cis-12 nd the cis-9, trns-11 isomer ws dministered; in contrst, Terpstr [23] reported in review tht mny studies did not show ny significnt effect of CLA on plsm cholesterol concentrtions or on LDL-cholesterol concentrtions. In this respect, we ssume tht cholesterol concentrtions my e relted to the isomers in the CLA mixture. In this study, serum triglyceride nd cholesterol concentrtions my hve een influenced y iopolymers (pectin nd chitosn) cting s dietry fiers. Dietry fiers my interfere with sorption of lipid droplets (with cholesterol) into the villi in the smll intestine; lterntively, they my interfere with the ility of lipse to ccess the lipids contined within them. Beysserit et l. [12] reported tht the ility of dietry fiers to reduce cholesterol digestion nd/or sorption through this mechnism depends on their ility to promote droplet ggregtion or to dsor lipid droplet on the surfce, which strongly depends on the electricl chrge, moleculr weight nd structure of fiers. In this study, lipid droplets in the diets were encpsulted with iopolymers (see Figure 6). Furthermore, the residul lipids in the smll intestines of mice in the dietry CLA nd iopolymer groups (HFC nd HFCB) were higher thn those in the control group (HF) (Figure 6). These results indicte tht iopolymer encpsultion of lipids could decrese lipid digestion; thus, triglyceride nd cholesterol concentrtions in serum were decresed. Mun et l. [24] reported tht ctionic chitosn cn form polymer coting round lipid droplets tht re stilized y nionic surfce-ctive mterils, such s phospholipids nd ile slt. In this study, iopolymer encpsultion round lipid droplets my hve inhiited the ility of lipse or ile slt to ccess the surfce of the lipid droplets, resulting in decresed digestion or sorption of triglycerides nd cholesterol. Conversely, high-ft diet supplemented with CLA (HFC) did not reduce lood cholesterol concentrtions in this study. This result indictes tht triglyceride nd cholesterol reduction is minly influenced y dietry supplementtion with iopolymers nd tht dietry CLA is less influentil. Thus, iopolymer supplementtion cn improve the effect of dietry CLA y inhiiting lipids, including triglycerides nd cholesterol. However, our preliminry study (dt not shown) reveled tht smll mounts of dietry CLA with iopolymers (less thn 0.1%) did not decrese serum cholesterol nd triglyceride concentrtions, lthough dietry CLA did reduce the dipose tissue weight of mice. Therefore, we elieve the mounts of oth dietry CLA nd iopolymers ply n importnt role in lipid digestion nd sorption. Erlier studies hve suggested tht energy sources hve lrge influence on physicl ctivity nd weight gin. Wells et l. [25] reported tht ehvior ws different fter ft nd crohydrte ingestion; ft seems to induce more restful feelings thn crohydrtes. Horton et l. [5] reported tht greter proportion of excess energy is stored during ft overfeeding (pproximtely 90% 95%) thn n equivlent mount of crohydrte overfeeding (pproximtely 75% 85%). In this study, voluntry trvel distnce s mesure of physicl ctivity ws greter in the low-ft (5%) diet group (LF) thn tht in the high-ft (20%) diet group (HF). This my e ecuse the low-ft diet in this study contined more crohydrte sources thn the high-ft diet groups under the sme clorie conditions. The
Int. J. Mol. Sci. 2013, 14 6856 high-ft diet with CLA nd iopolymers groups (HFC nd HFCB) showed n increse in voluntry trvel distnce. This finding indictes tht dietry CLA cn influence physicl ctivity without the intervention of iopolymers. It hs een proposed tht CLA reduces diposity y elevting energy expenditure vi incresed sl metolic rte, thermogenesis or lipid oxidtion in nimls [26]. Mizunoy et l. [27] lso suggested tht oxygen consumption incresed in mice fed diet contining 1% CLA for eight weeks. In this study, the dietry CLA groups (HFC nd HFCB) showed incresed voluntry trvel distnce compred with tht in the other dietry groups (LF nd HF). This result my e one of the min cuses of the dipose tissue depletion effect y dietry CLA. However, in this study, dietry supplementtion with iopolymers did not hve significnt influence in voluntry trvel distnce s mesure of physicl ctivity. From the results of this study, we ssume tht dietry CLA nd iopolymers do not exert negtive effects on physicl ctivity in mice model. Figure 6. Confocl fluorescence imges showing smll intestinl contents fter stining with Nile red. All green colored imges represent stined lipids. In the present study, the viscosity of smll intestinl contents my e ssocited with lipid digestion nd sorption in the smll intestine; in ddition, differences in the viscosity of smll intestinl contents would cuse impirment in the hydrolysis nd soluiliztion of lipids [28]. Meyer nd Doty [29] lso reported tht high viscosity of the contents of the smll intestine my dely lipid digestion, promoting sorption in the more distl prt of the smll intestine. An increse in solution viscosity is responsile for decrese in the trnsit time of ingested food in the gstrointestinl trct. Therefore, n increse in solution viscosity cuses decrese in the trnsport rte of lipid digestion products present within micelles to the gstrointestinl wlls. Thus, n increse in viscosity resulting from dietry CLA with iopolymers cuses reduction in the digestion nd sorption rte of lipids. In generl, the ility of solule dietry fiers to increse solution viscosity, retrd moleculr diffusion nd ind to wter depends minly on their moleculr weight, conformtion nd interctions. Liron [11] reported tht dietry fiers cn lter the rekup nd colescence of lipid droplets in the stomch nd smll intestine, therey ltering the surfce re of lipids exposed to digestive enzymes. In Figure 6, undigested lipids in the smll intestines of our mice models pper to e higher in dietry
Int. J. Mol. Sci. 2013, 14 6857 high ft in the CLA nd iopolymers groups (HFC nd HFCB) thn tht in the other groups (LF nd HF). This finding indictes tht dietry CLA with iopolymers reduced lipid digestion or sorption. A lrge mount of undigested lipid content in the smll intestine due to the presence of dietry CLA nd iopolymers my e the min reson for the incresed viscosity of smll intestine contents in those groups. In this study, dipose tissue weight ws not significntly different etween the high-ft diet with CLA group (HFC) nd the high-ft diet with CLA nd iopolymers group (HFCB). Conversely, the concentrtions of triglyceride nd cholesterol were lower in the high ft with CLA nd iopolymers group (HFCB) thn those in the high ft with CLA group (HFC). This result indictes tht iopolymers re the min influence on serum triglyceride nd cholesterol concentrtions, wheres iopolymers my hve less influence on dipose tissue weight or ody weight. 4. Experimentl Section 4.1. Mterils CLA mixture used in this study contined 63% trns-10, cis-12 CLA, 26% cis-9, trns-11 CLA nd 7% other. CLA isomers (>96% pure) were purchsed from Mtrey (Plesnt Gp, PA, USA). Chitosn, pectin, Tween 20, cetic cid, sodium cette, sodium chloride, monosic sodium phosphte, disic sodium phosphte nd Nile red were purchsed from Sigm-Aldrich Chemicl Compny (St. Louis, MO, USA). Serum triglyceride, cholesterol nd glucose ssy kits were purchsed from Thermo Electron, Inc. (Louisville, CO, USA). 4.2. Animl nd Experimentl Diet Forty femle ICR mice (ge, 3 months; weight, 25 30 g) nd semi-purified powdered diet were purchsed from Hrln Tekld (Mdison, WI, USA). Isocloric diet formultions were designed y Hrln Tekld (Mdison, WI, USA). Animls were housed in individul, wire-ottomed cges in windowless room with 12-h light/drk cycle, under protocol pproved y the Animl Cre Committee of Konkuk University. Food nd wter were provided d liitum throughout the experiment. Fresh food ws given on dys 0, 2 nd 5. After 2 weeks of dpttion to the environment nd voluntry trvel distnce testing, 40 nimls were rndomly divided into 4 groups nd fed the following diets: 5% ft (T1: LF); 20% ft (T2: HF); 20% ft contining 1% CLA (T3: HFC); nd 20% ft contining 1% CLA with 0.5% iopolymer per kg (T4: HFCB). All groups were fed their respective diets for 6 weeks. The formultion of the experimentl diets nd tretment groups re listed in Tle 2.
Int. J. Mol. Sci. 2013, 14 6858 Items Tle 2. Experimentl diets nd tretment group. T1 5% ft T2 20% ft Tretments 1 T3 20% ft with 1% CLA T4 20% ft with 1% CLA +0.1% iopolymers Csein 202 249.50 247.01 247.01 L-cystine 3.03 3.69 3.65 3.65 Corn strch 409.96 158.82 157.21 157.21 Mltodextrin 133 151.50 149.99 149.99 Sucrose 101 123.20 121.97 121.97 Soyen oil 50.50 202 199.98 199.98 Cellulose 50.50 50.50 50 45 Minerl Mix 35.35 43 42.57 42.57 Vitmin Mix 10.10 12.32 12.20 12.20 Clcium Phosphte Disic 2.02 2.42 2.40 2.40 Choline Bitrtrte 2.53 3.03 3.00 3.00 TBHQ (ntioxidnt) 0.01 0.02 0.02 0.02 CLA 0 0 10 10 Biopolymers 0 0 0 5 Totl 1,000 1,000 1,000 1,000 1 Different ft contents nd the sme totl clorie mong the diets. Isocloric diets designed y Hrln Tekld (Mdison, WI, USA). CLA, conjugted linoleic cid; TBHQ, tert-butylhydroquinone. 4.3. Biopolymer Encpsultion Preprtion Ten mss percentge of chitosn ws dissolved in cette uffer solutions (100 mm cetic cid: sodium cette, ph 3.0, 0 150 mm NCl). Ten mss percentge of pectin ws dissolved in phosphte uffer solutions (2 M monosic sodium phosphte nd 2 M disic sodium phosphte, ph 7.0). These solutions were stirred for 12 h nd then mixed for 3 h using mgnetic stirrer. During mixing, 1 ml of Tween 20 (0.1%, ph 7.6) ws dded dropwise to reduce surfce tension nd enhnce formtion of encpsultion. Biopolymer encpsultion ws prepred y mixing finl volume of 10 wt% iopolymer solution nd CLA together for 1 h using io-homogenizer. The mixture ws continuously stirred for 15 min using power ultrsound t frequency of 10 MHz, nd this ws then mixed with the experimentl diets (finl volume: 1% CLA mixed with 0.5% iopolymers in diet). This process ws imed t developing coting lyer round the lipophilic CLA. Encpsultion of iopolymers nd CLA solution ws confirmed using confocl microscopy (Figure 6). 4.4. Food Intke, Body Weight nd Adipose Tissue Weight Food intke nd ody weight gin were mesured weekly, nd dipose tissues were weighed fter CO 2 sphyxition scrifice. 4.5. Leptin, Adiponectin, Triglyceride, Cholesterol nd Serum Glucose Concentrtions Blood ws collected y crdic vessel leeding from nesthetized mice. Serum ws otined y centrifugtion t 3,000 g for 15 min t 4 C. Serum leptin nd diponectin concentrtions were
Int. J. Mol. Sci. 2013, 14 6859 mesured using ELISA kit (R & D Systems, Inc., Minnepolis, MN, USA) techniques, s specified y the mnufcturers. Serum triglyceride, cholesterol nd glucose concentrtions were mesured using the enzymtic ssy kits (Thermo Electron, Inc., Louisville, CO, USA), s per the mnufcturers instructions. 4.6. Voluntry Trvel Distnce Voluntry trvel distnce s mesure of physicl ctivity ws determined using wheel running systems. Mice were plced in n individul cse (35 20 15 cm) contining running wheel with dimeter of 10 cm under 12-h light/drk cycle. After period of fmiliriztion (1 week), mice were llowed to run freely on the wheels. Dt were collected dily for period of 6 weeks. Voluntry trvel distnces were clculted through the use of the Ntionl Instruments Compct-DAQ, n NI 9411 module nd custom-designed LVIEW progrm, which converted wheel revolution dt to dily voluntry trvel distnce. 4.7. Viscosity of Smll Intestinl Contents Smll intestinl contents were collected y gentle finger stripping. The smll intestinl contents were pooled per tue. The viscosity of smll intestinl contents ws determined y the modified method of Turi et l. [30]. The viscosity ws mesured using rottionl rheometer (Physic MCR 300, Stuttgrt, Germny) in system of coxil cylinders (25 mm in dimeter). A totl of 200 µl of smll intestinl contents ws plced on temperture control plte (37 C) nd sher stress nd sher rte were then mesured for 1 min. 4.8. Confocl Lser Scnning Microscopy Smll intestinl contents were collected y gently finger stripping. The smll intestinl contents nd iopolymer encpsultion were nlyzed through confocl microscopy. A confocl scnning fluorescence microscope (Crl Zeiss, LSM 5 Live, GmH, Jen, Germny) with 20 ojective lens ws used to cpture confocl imges. Nile red ( lipid fluorescent dye) ws excited with 488-nm rgon lser line. The fluorescence emitted from the smple ws monitored using fluorescence detector (543 nm) with pinhole size of 150 µm. The resulting imges consisted of 512 512 pixels, with pixel size of 414 nm nd pixel dwell time of 5 s. 4.9. Sttistics The influences of dietry CLA nd iopolymer encpsultion on lipid metolism in mice fed high- nd low-ft-fed diets were nlyzed using SAS softwre (SAS Inst. Inc., Cry, NC, USA, 2001) y the generlized liner model procedure. The Student-Newmn-Keuls multiple rnge test ws used to compre differences etween mens. 5. Conclusions From the results of this study, we conclude tht the increse in the voluntry trvel distnce s mesure of physicl ctivity resulting from dietry supplementtion with CLA my e one of the min
Int. J. Mol. Sci. 2013, 14 6860 resons for lipid depletion. Groups of mice fed high-ft diet supplemented with CLA nd iopolymers showed higher viscosity of smll intestinl contents thn the controls. This increse in viscosity my e one of the min resons for triglyceride nd cholesterol depletion. Thus, we ssume tht the lipid depletion effect of CLA my e ccelerted y the ddition of iopolymers. However, the nture of the reltionship etween incresed physicl ctivity nd dietry CLA remins to e elucidted. The effect of CLA on lipid metolism could e influenced to lrge degree y the mixture of CLA isomers, CLA contents (or purity), dietry period or sujects. Therefore, future studies re needed to investigte how dietry CLA influences physicl ctivity in niml models nd how dietry supplementtion with iopolymers is relted to chnges in lipid metolism. The nture of the interction etween CLA nd iopolymers (e.g., encpsultion efficiency nd CLA encpsultion rte) lso requires further investigtion. Acknowledgments This pper ws supported y the SMART Reserch Professor Progrm of Konkuk University. Conflict of Interest The uthors declre no conflict of interest. References 1. Pesctello, L.S.; VnHeest, J.L. Physicl ctivity medites helthier ody weight in the presence of oesity. Br. J. Sports Med. 2000, 34, 86 93. 2. Westerterp-Plnteng, M.S.; Lejeune, M.P.G.M.; Kovcs, E.M.R. Body weight loss nd weight mintennce in reltion to hitul cffeine intke nd green te supplementtion. Oesity 2005, 13, 1195 1204. 3. Rosenum, M.; Leiel, R.L. The physiology of ody weight regultion: Relevnce to the etiology of oesity in children. Peditrics 1998, 101, 525 539. 4. Hill, J.O.; Melnson, E.L.; Wytt, H.T. Dietry ft intke nd regultion of energy lnce: Implictions for oesity. J. Nutr. 2000, 130, 284S 288S. 5. Horton, T.J.; Drougs, H.; Brchey, A.; Reed, G.W.; Peters, J.C.; Hill, J.O. Ft nd crohydrte overfeeding in humns: Different effects on energy storge. Am. J. Clin. Nutr. 1995, 62, 19 29. 6. Prk, Y.; Alright, K.; Liu, W.; Storkson, J.; Cook, M.; Priz, M. Effect of conjugted linoleic cid on ody composition in mice. Lipids 1997, 32, 853 858. 7. Hur, S.J.; Whitcom, F.; Rhee, S.; Y.P.; Good, D.J.; Prk, Y. Effects of trns-10, cis-12 conjugted linoleic cid on ody composition in geneticlly oese mice. J. Med. Food 2009, 12, 56 63. 8. Choi, J.S.; Jung, M.H.; Prk, H.S.; Song, J. Effect of conjugted linoleic cid isomers on insulin resistnce nd mrna levels of genes regulting energy metolism in high-ft fed rts. Nutrition 2004, 20, 1008 1017. 9. Ngo, K.; Wng, Y.M.; Inoue, N.; Hn, S.Y.; Bung, Y.; Nod, T.; Koud, N.; Okmtsu, H.; Yngit, T. The 10trns, 12cis isomer of conjugted linoleic cid promotes energy metolism in OLETF rts. Nutrition 2003, 19, 652 656.
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