Research Article Oral Administration of Alkylglycerols Differentially Modulates High-Fat Diet-Induced Obesity and Insulin Resistance in Mice

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1 Evidence-Bsed Complementry nd Alterntive Medicine Volume 2013, Article ID , 11 pges Reserch Article Orl Administrtion of Alkylglycerols Differentilly Modultes High-Ft Diet-Induced Obesity nd Insulin Resistnce in Mice Mingshun Zhng, 1,2 Shun Sun, 3 Ning Tng, 1 Wei Ci, 1,4 nd Linxi Qin 1,4 1 Xinhu Hospitl, Shnghi Institute for Peditric Reserch, Shnghi Jio Tong University, School of Medicine, Shnghi , Chin 2 Deprtment of Immunology, Nnjing Medicl University, Nnjing , Chin 3 Fudn Children s Hospitl, Fudn University, School of Medicine, Shnghi , Chin 4 Shnghi Key Lbortory of Peditric Gstroenterology nd Nutrition, Shnghi , Chin Correspondence should be ddressed to Wei Ci; ciw204@yhoo.com.cn nd Linxi Qin; microtuble@gmil.com Received 1 Jnury 2013; Accepted 5 June 2013 Acdemic Editor: Wei Ji Copyright 2013 Mingshun Zhng et l. This is n open ccess rticle distributed under the Cretive Commons Attribution License, which permits unrestricted use, distribution, nd reproduction in ny medium, provided the originl work is properly cited. Alkylglycerols (AKGs) from shrk liver oil (SLO) were demonstrted to hve strong potency to stimulte immune response. However, no study hs been conducted on the effects of AKGs on diet-induced obesity nd metbolic inflmmtory disorder. The purpose of the present study ws to investigte the effect of two AKGs isoforms on obesity nd insulin resistnce in mice fed highft (HF) diet. Forty-eight C57BL/6 mice were divided into norml, HF, HF +20mg/kg selchyl lcohol (SA), HF mg/kg SA, HF +20mg/kg btyl lcohol (BA), nd HF mg/kg BA groups. Body weight, fsting glucose, lipids, insulin nd leptin levels, serum IL-1β, ndtnf-α levels were compred mong different groups. Our results showed tht high-dose SA decresed body weight, serum triglyceride, cholesterol, fsting glucose level, insulin level, nd serum leptin level of the HF fed mice, while highdose BA incresed fsting insulin level of the HF fed mice. Pretretment of primry dipocytes with 10 μm SA or BA differentilly modultes LPS-medited MAPK nd NF-κB signling. Our study demonstrted tht orl dministrtion of AKGs hs differentil effects on HF-induced obesity nd metbolic inflmmtory disorder in mice. 1. Introduction Obesity hs become common public helth issue with cluster of metbolic bnormlities. The incidence of obesityrelted chronic diseses is incresing rpidly worldwide [1]. Evidence hs ccumulted indicting tht obesity is closely ssocited with stte of systemtic, low-grde inflmmtion chrcterized by ctivtion of inflmmtory signling pthwys nd bnorml cytokine production in dipose tissue [2, 3]. The cytokines produced by dipocytes include severl inflmmtory mrkers such s interleukin (IL)-6, tumor necrosis fctor (TNF)-α, nd monocyte chemottrctnt protein (MCP)-1 [4]. These cytokines re elevted in ptients with obesity nd insulin resistnce nd re highly ssocited with the development of crdiovsculr diseses nd type 2 dibetes mellitus. Recently, dietry supplements hve been used for prevention of obesity nd dibetes mellitus due to their high complince nd low toxicity. Shrk liver oil (SLO), wellknown dietry supplement, contins lkylglycerols (AKGs), squlene, nd essentil ftty cids [5]. It hs recently been shown tht SLO hs vrious phrmcologicl benefits such s chemoprotective properties ginst rective oxygen species s well s nti-inflmmtory, ntibcteril, ntifungl, nd nticncer potency [6]. AKGs, the mjor component of SLO, re glycerol ether lipids tht hve structurl chrcteristics of n ether linkge between ftty cid nd α-position of the glycerol bckbone. According to the ftty cid chin length nd the number of double bonds, severl derivtives of AKGs hve been identified. They include such substnces s btyl lcohol (BA), chimyl lcohol (CA), nd selchyl lcohol (SA) [7]. SA, the predominnt component of bioctive AKGs in the

2 2 Evidence-Bsed Complementry nd Alterntive Medicine SLO (ccounting for 59.4%), contins n unsturted bond in the long hydrocrbon chin (18C:1). CA nd BA, which re sturted in their hydrocrbon chins (16C:0 CA, 18C:0 BA), ccount for minor proportion of SLO (9.1% CA, 2.8% BA) [6]. AKGs re lso found in immune orgns such s bone mrrow nd spleen, indicting their importnt role in humn immune ctivity [8]. AKGs minly function by stimulting immune response to enhnce the humn defense ginst inflmmtion [9]. AKGs cn lso be pplied to tret leukemi nd solid tumor s well [10]. It ws demonstrted tht AKGs cn inhibit the growth, vsculriztion, nd dissemintion of lung crcinom tumors in mice [11, 12]. The ntidibetic effects of vrious bioctive food components hve gined widespred ttention. However, it ws lso demonstrted tht some nutrients such s selenium hve side-effect on energy metbolism if they re supplemented inppropritely [13]. AKGs hve been shown to hve cpbility of ctivting cytotoxic mcrophges leding to n enhnced phgocytosis nd elevting Th-1 cytokines such s TNF-α which re required for mcrophge ctivtion [14]. Adipose tissue mcrophges ply key role in obesityinduced inflmmtion nd insulin resistnce [15]. However, no study hs been conducted on the effects of AKGs on dietinduced obesity nd metbolic inflmmtory disorder. It is interesting to explore how AKGs ffect energy metbolism if consumed dily s nutrition supplement. Therefore, we exmined the effect of AKGs on lipopolyscchride- (LPS-) medited insulin resistnce nd induction of inflmmtory genes in high-ft (HF) fed mice. 2. Mterils nd Methods 2.1. Chemicls. SA ws purchsed from NIKKO Chemicls (Tokyo, Jpn). BA ws purchsed from Bchem (Bubendorf, Switzerlnd). Escherichi coli LPS 0111:B6 ws purchsed from Sigm-Aldrich (St. Louis, MO). Glucose, cholesterol, nd triglyceride kits were obtined from Kinghwk Phrmceuticl (Beijing, Chin). Insulin, leptin, IL-1β, nd TNF- α ELISA kits were purchsed from R&D systems (Minnepolis, MN). Antiphospho- (Thr183/Tyr185) nd totl JNK, ntiphospho- (Thr202/Tyr204) nd totl ERK, nd nti-iκbα were purchsed from Snt Cruz (Snt Cruz, CA). All other chemicl regents used in the present study were of nlyticl grde Animls nd Fcilities. The study ws pproved by the Animl Ethics Committee of Xinhu Hospitl. Forty-eight, 4-week old mle C57BL/6 mice were purchsed from SLAC Lbortories (Shnghi, Chin). All mice were housed in stinless steel cges with bedding (6 mice/cge). Sufficient bedding ws used to keep mice dry nd clen. All the mice were exposed to 12-hour light nd drk cycle. Frequent bedding chnges nd cge clening were performed s often s necessry Animl Study Design. After rrivl, mice were cclimtized for 4 dys. After cclimttion, forty-eight mice were rndomly divided into six groups of 8 mice ech. Both norml chow nd high-ft diets were purchsed from Shnghi Slc Lbortory Animl Co., Ltd. Norml chow diets contined 20.5% crude protein, 4.62% crude ft, 52.5% nitrogen-free extrct, nd 4.35% crude fibers (totl clories 3.45 Kcl/g, 12% clories in ft). High-ft diets contined 18.8% crude protein, 16.2% crude ft, 45.2% nitrogen-free extrct, nd 3.98% crude fibers (totl clories 3.79 Kcl/g, 38% clories in ft) [16]. For 8 weeks, groups 1 nd 2 received the norml diets (ND) nd high-ft diets (HF), respectively; groups 3 nd 4 were fed the HF supplemented with 20 nd 200 mg/kg SA, respectively; groups 5 nd 6 were fed the HF supplemented with 20 nd200mg/kgba,respectively.bodyweightwsmonitored weekly. At the end of the experiment, blood smples were collected fter overnight fsting. Following 4 dys recovery, llgroupswerefstedfor5hoursndthenchllengedwith 100 ng LPS intrperitonelly. After 2 hours, nimls were then euthnized nd blood smples, liver, nd epididyml ft were collected. Liver tissues nd viscerl dipose were immeditely weighted fter removl [17]. Serum ws isolted by centrifugtion t 1500 g t 4 C for 10 min nd stored t 80 Cuntilitwsusedforbloodbiochemiclssys Culturing of Primry Adipocytes. Abdominl white dipose tissue ws obtined from 4- to 5-week-old, wildtype mice. After blood wshing, the dipose tissues were mincednddigestedwith1mg/mlcollgensetypei(sigm- Aldrich, St. Louis, MO) for 30 min t 37 C. Cells were filtered through200-μm pore size nylon meshes. The stroml vsculr cells (SVCs) were seprted from dipocytes by centrifugtion nd wshed with DMEM (Invitrogen, Crlsbd, CA) supplemented with 10% fetl bovine serum (FBS). SVCs were plted nd propgted to confluence in DMEM supplemented with 10% FBS, 50 μg/ml streptomycin, nd 50 U/mL penicillin [18]. After ttchment, the medium ws replced by induction medium contining 10 μg/ml insulin (INS), 1 μm dexmethsone (DEX), nd 0.5 mm 3-isobutyl-1- methylxnthine (MIX) with 10% FBS nd continued differentition for 12 dys. On dy 12, cultures were pretreted with DMSO vehicle, or different concentrtions of SA or BA for 24hrs,ndthentretedwith10μg/L LPS for 6 hrs Immunoblotting Anlysis. Following tretment, cultures were hrvested nd protein ws extrcted with RIPA buffer. Immunoblotting nlysis ws performed s described previously [19] Biochemicl Anlysis. Concentrtions of insulin, leptin, IL-1β, nd TNF-α were mesured using ELISA kit. Glucose, totl cholesterol, nd triglyceride were tested using enzymtic methods. Homeosttic Model Assessment-Insulin Resistnce (HOMA-IR) ws clculted from glucose nd insulin concentrtions (fsting glucose (mmol/l) fsting insulin (μu/ml)/22.5) [20] Sttisticl Anlysis. Dt re shown s mens with their stndrd errors. Sttisticl significnce ws evluted using one-wy ANOVA followed by Duncn s multiple rnge test. P vlue < 0.05 ws considered sttisticlly significnt.

3 Evidence-Bsed Complementry nd Alterntive Medicine 3 3. Results 3.1. Effects of AKGs Diets on Body nd Orgn Weights. Dily food intke during the experimentl period ws not significntly different mong groups. No chnges of end-point body weight nd net weight gin were observed between HF diet group nd low-dose (20 mg/kg) AKGs (SA or BA) supplemented groups during the 60-dy period. High-dose SA (200 mg/kg) supplementtion significntly decresed the end-point body weight nd net weight gin of the HF fed mice during the 60-dy dietry intervention. Weights of epididyml white dipose tissue nd liver were compred mong the different dietry tretments. Epididyml ft ws significntly decresed s percent body weight in mice tht received 200 mg/kg SA supplementtion (Tble 1) Effects of AKGs Diets on Serum Triglyceride nd Cholesterol. There ws significnt (110%) increse in the serum triglyceride level of the HF group compred with the ND group, wheres 25% decrese of serum triglyceride ws observed in the 200 mg/kg SA group reltive to the HF group (P < 0.01) (Figure1()). HF feeding cused significnt (50%) increse in the totl cholesterol level. 200 mg/kg SA tretment; however, hd reduced the serum cholesterol level by 30% s compred to the HF group (P < 0.001) (Figure 1(b)). There ws no significnt difference in triglyceride or cholesterol level between the HF group nd the HF plus low-dose SA group. No significnt chnge in triglyceride or cholesterol level ws observed in HF plus BA diet group s compred with HF group. Cholesterol (mmol/l) TG (mmol/l) ND HF P < 0.01 HF + 20 mg/kgsa () Pre-LPS HF mg/kgsa Pre-LPS P < HF + 20 mg/kgba HF mg/kgba 3.3. Effects of AKGs Diets on Glucose, Insulin, nd HOMA-IR. To investigte the impct of different AKGs supplemented HF diets in comprison with HF diet on glucose metbolism, we exmined blood glucose concentrtions before nd fter LPS chllenge in different dietry groups. HF diet group hd significntly higher fsting blood glucose concentrtion s compred to ND diet group (9.84 mmol/l versus 7.62 mmol/l; P < 0.001)(Tble2). Fsting blood glucose ws significntly lower in mice fed HF diet plus 20 or 200 mg/kg SA thn tht in mice fed HF diet (P < 0.001). After intrperitonel injection of LPS, drop of blood glucose concentrtion ws observed in ll groups. HF diet plus 200 mg/kg SA cused lower blood glucose concentrtion fter LPS chllenge s compred to HF diet group (5.05 mmol/l versus 5.71 mmol/l; P < 0.05)(Tble 2). The impct of different forms of AKGs on insulin resistnce induced by HF diet ws lso ssessed. The plsm insulin concentrtions were exmined before nd fter LPS chllenge in different dietry groups. As expected, HF diet group hd significntly higher fsting insulin concentrtions s compred to ND diet group (27.81 μiu/ml versus μiu/ml; P < 0.001) (Tble 2). It ws noted tht fsting insulin concentrtion ws decresed in mice fed HF diet plus 200 mg/kg SA reltive to mice fed HF diet (20.92 μiu/ml versus μiu/ml; P < 0.05), wheres insulin concentrtion ws incresed in mice fed HF diet plus 200 mg/kg BA (P < 0.05). After intrperitonel injection of ND HF HF + 20 mg/kgsa (b) Figure 1: The effect of selchyl lcohol (SA) or btyl lcohol (BA) supplementtion on serum triglycerides () nd cholesterol (b). Dt were presented s men ± SE. Significnt differences were tested with two-wy ANOVA. Only significnt comprisons re presented. LPS, mice fed HF plus 200 mg/kg SA showed lower insulin increse compred to mice fed HF diet (22.92 μiu/ml versus μiu/ml; P < 0.05). No effects of low-dose AKGs (SA or BA) supplementtion were observed on fsting insulin concentrtions t pre- nd post-lps chllenge (Tble 2). The HOMA-IR score ws clculted from fsting blood glucosendinsulinconcentrtiontossesswhetherakgs diet protected mice from insulin resistnce. In fct, mice tht received 20 or 200 mg/kg SA supplementtion hd significntly lower HOMA-IR scores (P < 0.001), however, mice received 200 mg/kg BA supplementtion hd significntly higher HOMA-IR scores s compred to mice tht received HF diet t pre-lps chllenge (P < 0.05). After intrperitonel injection of LPS, only 200 mg/kg SA supplementtion showed HF mg/kgsa HF + 20 mg/kgba HF mg/kgba

4 4 Evidence-Bsed Complementry nd Alterntive Medicine Tble 1: The effect of selchyl lcohol (SA) or btyl lcohol (BA) supplementtion on body weights, tissue weights, nd food consumption. P vlue 6 P vlue P vlue Item ND HF HF + 20 mg/kg SA HF mg/kg SA HF + 20 mg/kg BA HF mg/kg BA (HF) (SA) (BA) Initil body wt 2 (g) ± ± ± ± ± ± End-point body wt 3 (g) ± ± ± ± ± ± 0.39 <0.001 < Netweightgin 4 (g) ± ± ± ± ± ± 0.54 <0.001 < Epididyml ft 3 (%) 1.76 ± ± ± ± ± ± 0.07 < Liver weight 3 (%) 3.86 ± ± ± ± ± ± 0.03 < Food intke (g/mouse/dy) 2.80 ± ± ± ± ± ± ND: norml diet; HF: high-ft diet; SA: selchyl lcohol; BA: btyl lcohol. 2 Mesured before dietry intervention. 3 Mesured fter 8-week feeding period. 4 Difference between end-point body weight nd initil body weight. 5 Vlues re men ± SEM. Mens with mrk ( ) in HF + 20 mg/kg SA or HF mg/kg SA group differ significntly from the HF diet group. Mens with mrk ( )inhf+20mg/kgbaorhf+ 200 mg/kg BA group differ significntly from the HF diet group. 6 P vlue (HF) indictes the effect of HF tretment. P vlue (SA) indictes the effect of different doses of SA supplements on HF fed mice. P vlue (BA) indictes the effect of different doses of BA supplements on HF fed mice.

5 Evidence-Bsed Complementry nd Alterntive Medicine 5 significnt protective effect on insulin resistnce which ws indicted by HOMA-IR scores (P < 0.005)(Tble 2). 3.4.EffectofAKGsonSerumCytokinesndLeptin. The proinflmmtory cytokines such s IL-1β nd TNF-α were elevted fter LPS chllenge. Interestingly, high-dose SA or BA showed differentil effects on serum IL-1β nd TNF-α production fter 100 ng LPS chllenge (Tble 3). 200 mg/kg SA supplementtion suppressed serum IL-1β nd TNF-α level induced by LPS chllenge (P < 0.05), wheres 200 mg/kg BA supplementtion significntly enhnced serum IL-1β nd TNF- α level induced by LPS chllenge (P < 0.05). However, there ws no significnt effect on IL-1β nd TNF-α response to LPS chllenge by low-dose AKGs (SA or BA) supplementtion. As for serum leptin, mice tht received HF diet hd significntly higher serum leptin concentrtion compred to those received ND diet (16.55 ng/ml versus 7.26 ng/ml; P < 0.05). We found tht serum leptin concentrtion ws significntlydecresedinmicethtreceived200mg/kgsa supplementtion s compred to mice tht received HF diet t pre-lps chllenge (13.57 ng/ml versus ng/ml; P < 0.05)(Tble3) Effect of AKGs on LPS-Medited MAPK nd NF-κB Activtion. Given the role of MAPK pthwy in inducing inflmmtory gene expression vi Toll-like-receptor- (TLR-) 4 ctivtion, we exmined the effects of AKGs on MAPK phosphoryltion. Pretretment of mice dipocyte cultures with 10 μm SA modestly decresed LPS-medited phosphoryltion of JNK nd ERK (Figure 2). However, pretretment of mice dipocyte cultures with 10 μm BA incresed LPS-medited phosphoryltion of JNK nd ERK. In the bsence of LPS, AKGs tretment did not enhnce the MAPK ctivtion. Becuse the ctivtion of NF-κB lso plys crucil role in the trnscriptionl ctivtion of inflmmtion-responsive genes, the effects of AKGs on NF- κb ctivtion were exmined by detecting IκBα degrdtion vi immunoblot. We found tht the pretretment with SA ttenuted IκBα degrdtionby LPS (Figure2). However, BA did not present effect on LPS-induced NF-κB ctivtion in our experiments. 4. Discussion SLO hs been widely used in the pst yers in the Scndinvin medicine becuse of its properties s immunity boosters nd remedy ginst rdition therpy nd cncer [21]. AKGs re the mjor components in SLO which could stimulte immunity both in vitro nd in vivo [22, 23]. Dily consumption of AKGs-rich SLO showed benefits to the immune system. Despite widespred intke of AKGs, sfety studies on AKGs extrct were poor. The cute nd repeted (28 dys) orl toxicity hs been evluted for orl AKGs dministrtion in rts t doses of 200 nd 1000 times the mximum recommended dose in humns [24]. In tht study, AKGs dministrtion showed no dverse effects on mortlity t either cute or subchronic dose. However, the correltion between long-term AKGs supplement nd HFinduced obesity hs not been shown. In previous studies, AKGs were demonstrted to hve potency to ctivte cytotoxic mcrophges nd increse humorl immune response [22]. AKGs could lso stimulte the IL-12 nd IFN-gmm production nd elicit Th1 response [25, 26]. As we know, dipose infiltrted mcrophge nd secreted cytokines ply importnt roles in obesity nd insulin resistnce [27 29]. Thus, the potency of AKGs to stimulte immunity spurred our interest to exmine the AKGs effect on HF-induced obesity nd insulin resistnce. Studies hve shown tht HF diets induce dipose tissue inflmmtion nd stimulte TLR-4 expression [30]. TLR- 4, subclss of the TLR fmily, plys criticl role in ctivting innte immune nd inflmmtion response in mmmls by recognizing bcteril LPS [31, 32]. Activtion of TLR-4 in dipocytes leds to the ctivtion of MAPK nd NF-κB signling pthwys, nd induction of mny inflmmtory cytokines [33, 34]. These cytokines re involved in inducing glucose intolernce, insulin resistnce, nd infiltrtion of mcrophges into dipose tissue. Recent study showed tht TLR-4 responds to nonbcteril lignds such s ftty cids [35 37]. It ws demonstrted tht sturted ftty cids such s luric cid nd plmitic cid re ble to induce cyclooxygense-2 (COX-2) expression; however, unsturted ftty cids such s docoshexenoic cid (DHA) nd eicospentenoic cid (EPA) re ble to inhibit sturted ftty cid-induced COX-2 expression [38]. In present study, we demonstrted tht AKGs with sturted chin incresed LPS-medited ctivtion of the MAPK signling, which could cuse the expression of inflmmtory genes nd insulin resistnce in dipocytes. AKGs with unsturted chin decresed LPS-meditedctivtionoftheMAPK ndnf-κb signling, thus meliorted insulin resistnce. However, both AKGs with sturted nd unsturted chin did not ctivte MAPK or NF-κB signling in the bsence of LPS. Accordingly, it ws indicted tht both AKGs with sturted chin nd unsturted chin modulted TLR-4 signling nd insulin response in the hyperinflmmtory environment such s high-ft diet feeding or LPS tretment but did not show direct effect in the hypoinflmmtory environment. This result ws lso noticed by Ocñ et l., who showed tht BA ctivted the expression of IL-1β nd IL-6 genes only in TNF-α-induced dipocytes but not in the nonstimulted cells [39]. Severl studies hve demonstrted tht different forms of AKGs hve differentil biologicl effects. The unsturted AKGs with 16 or 18 crbon lkyl chins showed strong nti-tumor nd ntimetstsis ctivities in mice model [40]. By contrst, the sturted AKGs with 16 or 18 crbon lkyl chins showed less ntitumor effect or even tumorpromoting ctivity. Previous studies lso demonstrted tht proinflmmtory responses were enhnced by most sturted ftty cids but reduced by most unsturted ftty cids. In metbolic experiments, rts fed sturted ftty cids hd higher tricylglycerol, cholesterol, nd low-density lipoprotein cholesterol, wheres rts fed unsturted ftty cids hd lower tricylglycerol, cholesterol, nd low-density lipoprotein cholesterol s compred with control rts [41, 42]. Moreover, the incresed rtio of P/S (polyunsturted/sturted) ftty

6 6 Evidence-Bsed Complementry nd Alterntive Medicine Tble 2: The effect of selchyl lcohol (SA) or btyl lcohol (BA) supplementtion on serum glucose, insulin, nd Homeosttic Model Assessment-Insulin Resistnce (HOMA-IR) before nd fter 100 ng lipopolyscchride (LPS) chllenge. P vlue 4 P vlue P vlue Item ND 2 HF HF + 20 mg/kg SA HF mg/kg SA HF + 20 mg/kg BA HF mg/kg BA (HF) (SA) (BA) Glucose (mmol/l) 7.62 ± ± ± ± ± ± 0.54 <0.001 < Insulin (μiu/ml) ± ± ± ± ± ± 2.03 < HOMA-IR 4.63 ± ± ± ± ± ± 1.17 <0.001 < Post-LPS Glucose (mmol/l) 6.19 ± ± ± ± ± ± Insulin (μiu/ml) ± ± ± ± ± ± 2.33 < HOMA-IR 5.71 ± ± ± ± ± ± Serum glucose nd insulin were mesured before LPS chllenge (Pre-LPS) nd 2 hours following 100 ng LPS chllenge (Post-LPS). 2 ND: norml diet; HF: high-ft diet; SA: selchyl lcohol; BA: btyl lcohol. 3 Vlues re men ± SEM. Mens with mrk ( ) in HF + 20 mg/kg SA or HF mg/kg SA group differ significntly from the HF diet group. Mens with mrk ( ) in HF + 20 mg/kg BA or HF mg/kg BA group differ significntly from the HF diet group. 4 P vlue (HF) indictes the effect of HF tretment. P vlue (SA) indictes the effect of different doses of SA supplements on HF fed mice. P vlue (BA) indictes the effect of different doses of BA supplements on HF fed mice.

7 Evidence-Bsed Complementry nd Alterntive Medicine 7 Tble 3: The effect of selchyl lcohol (SA) or btyl lcohol (BA) supplementtion on serum cytokines nd leptin before nd fter 100 ng lipopolyscchride (LPS)chllenge. P vlue 4 P vlue P vlue Item ND 2 HF HF + 20 mg/kg SA HF mg/kg SA HF + 20 mg/kg BA HF mg/kg BA (HF) (SA) (BA) IL-1β (pg/ml) 2.3 ± ± ± ± ± ± 1.37 < TNF-α (pg/ml) 4.83 ± ± ± ± ± ± Leptin (ng/ml) 7.26 ± ± ± ± ± ± 0.55 < Post-LPS IL-1β (pg/ml) ± ± ± ± ± ± 8.7 < TNF-α (pg/ml) 231 ± ± ± ± ± ± < <0.001 Leptin (ng/ml) 9.18 ± ± ± ± ± ± 0.68 < Serum cytokines nd leptin were mesured before LPS chllenge (Pre-LPS) nd 2 hours following 100 ng LPS chllenge (Post-LPS). 2 ND: norml diet; HF: high-ft diet; SA: selchyl lcohol; BA: btyl lcohol. 3 Vlues re men ± SEM. Mens with mrk ( ) in HF + 20 mg/kg SA or HF mg/kg SA group differ significntly from the HF diet group. Mens with mrk ( ) in HF + 20 mg/kg BA or HF mg/kg BA group differ significntly from the HF diet group. 4 P vlue (HF) indictes the effect of HF tretment. P vlue (SA) indictes the effect of different doses of SA supplements on HF fed mice. P vlue (BA) indictes the effect of different doses of BA supplements on HF fed mice.

8 8 Evidence-Bsed Complementry nd Alterntive Medicine p-jnk p-erk JNK ERK Rtio P-JNK/JNK DMSO b 10 μm 10 μm DMSO 10 μm 10 μm SA BA SA BA LPS ( ) LPS (+) c Rtio P-ERK/ERK DMSO b b 10 μm 10 μm DMSO 10 μm 10 μm SA BA SA BA LPS ( ) LPS (+) c () (b) IκBα β-actin Rtio IκBα/ctin DMSO b 10 μm 10 μm DMSO 10 μm 10 μm SA BA SA BA LPS ( ) LPS (+) (c) b Figure 2: The effect of selchyl lcohol (SA) or btyl lcohol (BA) on TLR-4 signling ctivted by LPS in mice primry dipocytes. Newly differentited cells were pretreted with DMSO vehicle ( )or10μmsaor10μmbafor24hndthentretedwith10μg/l LPS for 6 h. () Phosphoryltion of JNK (P-JNK-to-JNK rtio). (b) Phosphoryltion of ERK (P-ERK-to-ERK rtio). (c) Expression of IκBα (IκBα-to-ctin rtio). Levels of protein were mesured by western blotting. Dt re men of ± SE nd corrected for loding. Significnt differences between ech group re indicted by different letters. cids ws beneficil in depleting white dipose tissue ccumultion nd improved the metbolic sttus in diet-induced obese hmster [43]. The controlled clinicl trils hve lso indicted tht replcing sturted ft with unsturted ft ws more effective in lowering risk of metbolic disorder thn simply reducing totl ft consumption [44, 45]. Our studies showedthtakgshdthesimilreffectsonmetbolicnd inflmmtory sttus s free ftty cids in spite of their ether bond with glycerol. As we know, SLO nd humn brest milk re both good sources of AKGs, with unsturted forms s the predominnt components. In clinicl studies, SLO showed beneficil effect on lipid metbolism in ptients with hypertension [46]. Brest milk hs lso been demonstrted to be protective ginst the development of childhood overweight nd obesity [47]. Thus, we postulted tht the predominnt unsturtedakgsinslondbrestmilkwouldneutrlize the dverse effects of sturted AKGs on metbolism nd ply prominent role of preventing metbolic syndrome. Incresing studies hve indicted tht dietry supplementtion of oils extrcted from mrine species cn lter the metbolic nd immunologic sttus which is highly ssocited with the incidence of obesity nd type 2 dibetes. Different mrine oils hve distinct lipid profiles, which result in potentil differences in their effects on inflmmtion nd metbolism. Fish oil, the typicl mrine oil product, ws demonstrted to hve protective effect on LPSinduced inflmmtion nd insulin resistnce [17]. It ws lso shown tht dietry fish oil supplementtion could reduce body weight gin in HF-induced obese mice [48]. The nti-inflmmtion nd ntiobesity effects of fish oil were ttributed to its richness of (n 3) PUFA [49]. SLO, which ws lso rich of (n 3) PUFA,wsknowntocontinhigh

9 Evidence-Bsed Complementry nd Alterntive Medicine 9 proportion of squlene nd AKGs. Both squlene nd AKGs hve potent immunologicl ctivity, thus eliciting people s interest of investigting their impct on metbolic blnce. Squlene hs lredy been demonstrted to be ble to elevte the body weight nd serum cholesterol in high-ft fed hmsters due to its involvement in heptic cholesterol metbolism [50]. However, little is known bout the ssocition between AKGs intke nd metbolic ltertion. Our results showed tht AKGs differentilly modulted HF-induced obesity nd insulin resistnce in mice, which gve us indiction tht more ttentionshouldbepidtotheimpctoftheseimmunestimulting chemicls on metbolism. Actully, it hs lredy been noticed tht orl SLO intke hs dverse effect on liver function if supplemented t high dosge [51]. Therefore, it is importnt to use AKGs in niml experiment t the dosge comprble to tht of humn supplementtion for nutritionl purpose. The Biomre Immuno which is mnufctured by Hnkinttukku (Finlnd) contins 60 mg AKGs per cpsule. On the lbel, it recommends six cpsules dily, which equls to 7.2 mg AKGs/kg weight (60 mg 6/50 kg weight )providedthtmledultweighs 50 kg on verge. In the present study, AKGs t 20 mg/kg diet nd 200 mg/kg diet dietry supplementtion corresponded to 4 mg/kg weight (20 mg/kg diet kg diet /0.025 kg weight )nd 40 mg/kg weight (200 mg/kg diet kg diet /0.025 kg weight ), respectively, ssuming tht mle dult mouse weighs 25 g nd expends 5 g diet on verge. Thus, the mount of 20 mg/kg diet AKGs dietry supplement ws comprble to tht commonly consumed by SLO-supplement consumer. The results obtined from rodents showed tht the 20 mg/kg diet dietry AKGs supplementtion did not hve significnt effect on body weight but only showed minor effect on glucose metbolism. Nevertheless, 200 mg/kg diet dietry AKGs supplementtion showed obvious effects not only on body weight but lso on lipid, glucose metbolism, nd pro-inflmmtory cytokine production. Although these results were not directly pplicble to humns, they provided some implictions for individuls who routinely consume AKGs supplement or AKGs-rich SLO. Tken together, our results provided novel insights into differentil effects of sturted- nd unsturted-chin AKGs on dipose tissue inflmmtion, which suggested tht routine consumption of SLO consisting of different isoforms of AKGs is sfe t norml dosge nd shows blnced effect on obesity nd insulin resistnce. 5. Conclusion Collectively, these dt demonstrte tht the unsturted AKGs (SA) hve potency to decrese the HF-induced obesity t high dosge nd meliorte insulin resistnce t norml or high dosge. The sturted AKGs (BA) cn increse insulin resistnce t high dosge. Our dt lso suggest tht AKGs show differentil effects on LPS-induced inflmmtion in dipocytes. The effect of SLO rich for AKGs on dipose metbolism nd insulin response should be evluted in the future, to wrrnt supplementing SLO products t sfe dosge nd prevent its potentil hzrdous effects on metbolism. Abbrevitions AKGs: lkylglycerols SA: Selchyl lcohol BA: Btyl lcohol HF: High ft ND: Norml diets LPS: Lipopolyscchrides IL: Interleukin TNF: Tumor necrosis fctor SLO: Shrk liver oil SVCs: Stroml vsculr cells HOMA-IR: Model ssessment-insulin resistnce TLR: Toll-like-receptor PUFAs: Polyunsturted ftty cids. Acknowledgments ThisreserchwssupportedbyGrnts , , , nd from the Ntionl Nturl Science Foundtion of Chin (to Linxi Qin, Wei Ci, nd Shun Sun), Progrm for Innovtive Reserch Tem of Shnghi Municipl Eduction Commission (to Wei Ci), 11QA Hesper Foundtion of Shnghi Scientific Bureu (to Linxi Qin), 2010Y157 Junior Scientist Foundtion of Shnghi Helth Bureu (to Linxi Qin), nd 11DZ Shnghi Key Lbortory of Peditric Gstroenterology nd Nutrition (to Wei Ci). References [1] H. Tilg nd A. R. 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