Visceral fat accumulation determines postprandial lipemic response, lipid peroxidation, DNA damage, and endothelial dysfunction in nonobese Korean men

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1 Viscerl ft ccumultion determines postprndil lipemic response, lipid peroxidtion, DNA dmge, nd endothelil dysfunction in nonobese Koren men Yngsoo Jng,*, Oh Yoen Kim, H Jung Ryu, Ji Young Kim, Sng Hoon Song,** Jose M. Ordovs, nd Jong Ho Lee 1,, Division of Crdiology,* Crdiovsculr Genome Center, Yonsei Medicl Institute, Yonsei University, Seoul, Kore; Yonsei University Reserch Institute of Science for Aging, Yonsei University, Seoul, Kore; Deprtment of Food & Nutrition, Yonsei University, Seoul, Kore; Food Ingredient Division, Foods R&D Center, CJ Corp.,** Seoul, Kore; nd Nutrition nd Genomics Lbortory, JM-USDA-HNRCA, Tufts University, Boston, MA Abstrct Viscerl ft hs been ssocited with multiple crdiovsculr disese (CVD) risk fctors. The im of this study ws to identify nthropometricl mesures most closely ssocited with some well-known CVD risk fctors. Becuse most Asins t risk hve norml body mss index (BMI) ccording to Western stndrds, we studied helthy nonobese Koren mles (n 102; ge: yers, BMI: kg/m 2 ). Viscerl ft re (VFA) t the fourth lumbr vertebr ws ssocited with incresed postprndil triglyceride (TG) response (r 0.53, P 0.001) nd with plsm mlondildehyde (MDA) (r 0.36, P 0.01) nd PGF2 (r 0.24, P 0.05). When mtched for BMI nd ge, men with high VFA (HVFA) ( 100 cm 2 ; n 27) hd higher blood pressure (P 0.01), incresed consumption of cigrettes (P 0.01), nd lower rtio of energy expenditure to clorie intke (P 0.01) s compred with low VFA men ( 100 cm 2 ; n 27). Men with HVFA showed higher TG, glucose, nd insulin responses following ft nd orl glucose tolernce tests respectively higher plsm concentrtions of MDA (P 0.001), urinry PGF 2 (P 0.05), nd lymphocytes deoxyribonucleic cid til moments (P 0.01). Conversely, HVFA ws ssocited with lower testosterone, insulin-like growth fctor-1, nd brchil rtery flowmedited diltion (P 0.001). In conclusion, our dt indicte tht viscerl ft ccumultion, even in nonobese men, is mjor fctor contributing to incresed CVD risk. Jng, Y., O. Y. Kim, H. J. Ryu, J. Y. Kim, S. H. Song, J. M. Ordovs, nd J. H. Lee. Viscerl ft ccumultion determines postprndil lipemic response, lipid peroxidtion, DNA dmge, nd endothelil dysfunction in nonobese Koren men. J. Lipid Res : flow-medited diltion body ft distribu- Supplementry key words tion glucose tolernce The most recent figures bout the epidemic of obesity nd overweight in the US nd other industrilized countries re pplling. Thirty-four percent of US dults re considered overweight, nd n dditionl 31 percent re obese. The consequences of this re fr beyond the esthetics of the popultion. Being overweight or obese increses the risk of hypertension, hert disese, stroke, dibetes, nd some cncers. In the US lone, 300,000 people die ech yer due to obesity-relted cuses, mking it the second-leding cuse of deth fter smoking. Asins experience similr risk of obesity-relted diseses, but the problem is more insidious, s the risk threshold t which body mss index (BMI) ppers to trigger the disese my be much lower for Asins ( 23 kg/m 2 ) thn for white popultions (30 kg/m 2 ) (1). This my result from differences in body frme tht ffect the reltion between body ft nd BMI. The popultion of Kore, similr to other industrilized countries in Asi, is experiencing drmtic nd fst chnges in dietry nd physicl ctivity hbits tht re the driving force behind the increse in obesity nd obesity-relted diseses. Koren dults hve incresed the percent of clories from ft in their diets from 6% in 1969 to 19% in Despite the greter thn 3-fold increse in the dietry ft intke, their dily clorie intke (bout 1,950 kcl/d) nd BMI (bout 22.5 kg/m 2 ) hve chnged little over the sme period (2). However, these lifestyle chnges hve drmticlly ffected the centrl dipose tissue distribution pttern s reflected by the globl increse of wisthip rtio (WHR). Among Korens, the mortlity rte (per 100,000 of the popultion) s result of ischemic hert disese hs rpidly incresed from 6.8 in 1988 to 13.8 in Mnuscript received 29 My 2003 nd in revised form 22 August Published, JLR Ppers in Press, September 1, DOI /jlr.M JLR200 1 To whom correspondence should be ddressed. e-mil: jhleeb@yonsei.c.kr 2356 Journl of Lipid Reserch Volume 44, 2003 This rticle is vilble online t

2 1997 (3). The increse in crdiovsculr disese (CVD) rtes is prtilly relted to the rpid increse in the number of elderly subjects; however, the increse of ft intke in Koren prllels lso the disese trends (2, 3). These temporl chnges suggest tht the increse in ft intke nd centrl ft distribution my be prtilly responsible for the incresing CVD rtes in Kore nd other neighboring countries. Previous reserch hs shown tht dipose tissue distribution is more closely ssocited with risk of dibetes, hypertension, nd hyperlipidemi thn totl body ft or BMI (4). Thus, viscerl ft vlues bove cm 2 represent strong risk for these metbolic disturbnces (5, 6). Moreover, both obesity nd viscerl ft ccumultion hve been reported to be relted with incresed postprndil lipemi (7), n underestimted CVD risk fctor. However, it remins uncertin which mesure of obesity represents the best predictor of postprndil lipemi response. Moreover, other behviorl fctors, such s lcohol consumption, cigrette smoking, or poor eting ptterns such s excessive clorie or high ft intke, re lso significnt determinnts of postprndil lipemi, s well s lipid peroxidtion, nd deoxyribonucleic cid (DNA) dmge (8 13). The objectives of this study were to identify the nthropometricl fctor most closely ssocited with vribility on the postprndil lipid response to ft lod chllenge nd to investigte the reltionships between this vribility, lipid peroxides, nd DNA dmge of lymphocytes in groups of helthy nonobese men ctegorized ccording to the relevnt nthropometricl fctor emerging from our reserch. Our findings will contribute to better understnding of the metbolic ltertions nd behviorl fctors responsible for the rpid increse in CVD mortlity in Kore nd to provide guidnce for preventive nd therpeutic interventions. METHODS Subjects Study subjects were recruited from volunteers who responded to dvertisements for nutrition study conducted by the Clinicl Nutrition Reserch Tem t Yonsei University in All subjects hd norml glucose tolernce tests nd electrocrdiogrms. None of them ws tking ny mediction or hd clinicl evidence of CVD or cncer. Finlly, 102 helthy men were selected s study subjects. The rnges for ge nd BMI were yers nd kg/m 2, respectively. Written informed consent ws obtined from ll subjects nd the protocol ws pproved from the Ethicl Committee of the Yonsei University. Mel tolernce test A 6 h postprndil lipemi response test ws crried out strting t 8:30 AM fter n overnight fst of greter thn 12 h. A stndrdized test mel ws prepred in the metbolic wrd using common food items. It consisted of sndwich contining white bred, lettuce, hm, nd soyben oil-bsed myonnise. The energy content, clculted from the computerized dtbse Koren food-code bsed on food composition tbles from the Ntionl Rurl Living Science Institute (6th edition, 2000) in Kore, ws 608 kcl (2.54 mj), representing the verge clorie intke from trditionl brekfst. Ft represented 41.4% (28 g) of the clories, crbohydrtes mde up 45.4% (69 g), nd 13.2% of clories (20 g) were derived from protein. In contrst, the mcronutrient composition of the subjects usul diet ws tht of typicl diet with cooked refined rice, consumed by substntil number of Korens, tht is, bout 57% of energy from crbohydrte, 22% from ft, 16% from protein, nd 5% from others (minly lcohol). Anthropometricl nd blood pressure mesurements Body weight nd height were mesured in the morning, unclothed nd without shoes. BMI ws clculted s body weight in kilogrms divided by height in squre meters. Wist nd hip circumferences were combined into the WHR representing commonly used surrogte of body ft distribution. Blood pressure ws red from the left rm while subjects remined seted. An verge of three mesurements ws recorded for ech subject. Regionl ft nd muscle res We performed computerized tomogrphy (CT) scnning using Generl Electric (GE) High Speed Advntge 9800 scnner (Milwukee, WI) to mesure ft nd muscle res. Four cross-sectionl imges were obtined from ech subject. Two bdominl ones t the level of the first lumbr (L1) nd fourth lumbr (L4) vertebrs, one from the thigh (midwy between ptell nd pubis), nd one from the clf (t the most protruding re). Ech CT slice ws nlyzed for the cross-sectionl re of ft using density control progrm vilble in the stndrd GE computer softwre. Prmeters for totl bdominl ft density t the levels of L1 nd L4 were selected between the rnge of 150 nd 50 Hounsfield units (HU). Totl bdominl ft re ws divided into viscerl nd subcutneous ft res to clculte specific ft res. Prmeters for thigh nd clf muscle res were selected s between the rnge of 49 nd 100 HU nd for ft res between 150 nd 50 HU. Blood collection Venous blood smples were obtined from the forerm nd collected into EDTA-treted nd plin tubes during fsting (bseline), nd t 2 h, 3 h, 4 h, nd 6 h fter brekfst for ssessment of glucose, insulin, free ftty cids (FFAs), nd triglycerides (TGs). Tubes were immeditely covered with luminum foil nd plced on ice until they rrived t the nlyticl lbortory (within 1 3 h) nd were stored t 70 C. Subjects were sked to refrin from performing strenuous exercise or drinking lcoholic beverges 24 h prior to the ft tolernce test. They were lso instructed to void eting or drinking nything except wter during the test period. Serum lipid profile nd polipoprotein A-I nd B Fsting serum concentrtions of totl cholesterol nd TG were mesured using commercilly vilble kits on Hitchi 7150 Autonlyzer (Hitchi Ltd., Tokyo, Jpn). After precipittion of serum chylomicron, LDL, nd VLDL with dextrn sulfte-mgnesium, HDL left in the superntnt ws mesured by n enzymtic method. LDL cholesterol concentrtions were estimted indirectly using the Friedwld formul for subjects with serum TG 4.52 mmol/l (400 mg/ml), or otherwise directly using commercilly vilble kits on Hitchi 7150 Autonlyzer. Serum polipoprotein A-I (poa-i) nd pob were determined by turbidometry t 340 nm using specific nti-serum (Roche, Switzerlnd). Orl glucose tolernce test nd homeostsis model ssessment At different time thn the ft tolernce test, we crried out glucose lod test in order to investigte glucose tolernce. Ech subject received 75 g glucose solution fter n overnight fst. Jng et l. Viscerl ft, postprndil lipemi, nd lipid peroxidtion 2357

3 Venous specimens were collected before nd 30 min, 60 min, 90 min, nd 120 min fter the glucose lod test. We used criteri, newly developed nd modified by the Ntionl Dibetes Dt Group nd the World Helth Orgniztion Expert Committee on Dibetes Mellitus, to ctegorize subjects ccording to their glucose sttus (14). Glucose ws mesured by glucose oxidse method using the Beckmn Glucose Anlyzer (Beckmn Instruments, Irvine, CA). Insulin ws mesured by rdioimmuno ssys with commercil kits from Immuno Nucleo Corportion (Stillwter, MN). FFA ws nlyzed with Hitchi 7150 utonlyzer (Hitchi Ltd., Tokyo, Jpn). Ech response of glucose, insulin, nd FFA ws clculted with the re under ech response curve. Insulin resistnce (IR) ws lso clculted with the homeostsis model ssessment (HOMA) (15) using the following eqution: IR [fsting insulin ( U/ml) fsting glucose (mmol/l)]/22.5. Other mesurements Immunordiometric ssys (IRMAs) were used to mesure serum totl testosterone using RIA-mt 280 (Byk-Sngtec Dignostic, Germny) with Cost-A-Count totl testosterone. Serum insulin like growth fctor-1 (IGF-1) ws mesured using the IRMA kit from Dignostic System Lbortories. Serum leptin ws mesured using Pckrd Cobr II 5005 R-Counter with the humn leptin RIA kit from Linco. Urine collection nd 8-epi-prostglndin F 2 nd plsm mlondildehyde Urine ws collected fter 12 h fst in polyethylene bottles contining 1% butylted hydroxytoluene before blood collection. The tubes were immeditely covered with luminum foil nd stored t 70 C until extrction. Urinry 8-epi-prostglndin F 2 (8-epi-PGF 2 ) ws quntified with gs chromtogrphy (Hewlett Pckrd 6890, Wilmington, DE) nd mss selective detector (Hewlett Pckrd 3973), ccording to the modified method of Prtico, Lwson, nd Fitzgerld (16) nd Mori et l. (17). Urinry cretinines were determined by the lkline picrted (Jeffe) rection (18), nd urinry 8-epi-PGF 2 levels were expressed s picogrm per milligrm cretinine (pgl/mg of cretinine). Plsm mlondildehyde (MDA) ws ssyed ccording to the fluorometric method described by Buckinghm (19). Alkline comet ssy for DNA dmge For the comet ssy, 120 l whole blood ws mixed with 900 l PBS nd poured gently over 150 l lymphocyte seprtion solution (HISTOPAQUE-1077). After centrifugtion t 1,450 rpm for 4 min, lymphocytes were pipetted out nd trnsferred to nother tube. DNA dmge ws nlyzed bsiclly s described by Green et l. (20). All steps were performed under dimmed light nd the electrophoresis tnk ws covered with blck pper to void dditionl light-induced DNA dmge. Vsodilttion response of brchil rtery Using high-resolution ultrsound (GE Vigmed Ultrsound, Horten, Norwy), we ssessed brchil rteril vsorectivity to rective hyperemi (flow-medited diltion; FMD) nd sublingul nitroglycerin (nitroglycerin-medited diltion; NMD) with the method described by Kng et l. (21). A 10 MHz liner phsed rry ultrsound trnsducer (GE Vigmed Ultrsound, Horten, Norwy) ws used to imge the dominnt rm brnchil rtery longitudinlly 3 5 cm just bove the ntecubitl foss. All subjects rested in the supine position for 10 min in quiet room. The stright segment of the rtery ws chosen. After the depth nd gin setting were optimized to identify the vessel wll, lumen interfce nd bseline brchil rtery dimeter were obtined. Brchil rtery dimeter ws mesured from the nterior to the posterior interfce between the medi nd the dventiti nd determined t end distole on 2D imge. Rective hyperemi ws induced by infltion nd then defltion of pneumtic cuff plced round the forerm portion. The blood pressure cuff ws inflted to 250 mm Hg for 5 min, creting distl limb ischemi. After relese of the cuff, brchil rtery dimeter ws mesured within the first 15 s of rective hyperemi. FMD ws then used s mesure of endothelium-dependent vsodiltion. The brchil rtery ws llowed to return to the initil bseline level 10 min fter cuff relese. A further bseline brchil rtery dimeter ws obtined. A 0.6 mg of nitroglycerin ws then given sublingully, nd the brchil rtery dimeter ws then mesured for the ensuing 3 min. The NMD ws used s mesure of endotheliumindependent vsodiltion. The percent chnge in dimeter cused by rective hyperemi ws clculted by dividing the difference from bseline end-distolic dimeter by the bseline vlue (FMD%). The percent chnge in dimeter cused by nitroglycerin dministrtion ws lso clculted in the sme wy (NMD%). Blood pressure nd hert rte were mesured before the exmintion. All dt were clculted s n verge from four consecutive crdic cycles. Assessment of food intke nd physicl ctivity level Usul food intke ws ssessed with 24 h recll method nd semi-quntittive food frequency questionnire. Nutrient intke dt were clculted s men vlues from the sme dt bse s referred bove. Totl clorie expenditure (kcl/dy) ws clculted from ctivity ptterns including bsl metbolic rte, physicl ctivity for 24 h (22), nd specific dynmic ction of food. Bsl metbolic rte for ech subject ws clculted with the Hrris-Benedict eqution (23). Sttisticl nlysis We used SPSS version 11.0 for Windows (Sttisticl Pckge for the Socil Science, SPSS Inc., Chicgo, IL) for ll our sttisticl nlyses. Ech vrible ws exmined for norml distribution nd significntly skewed vribles were log trnsformed. For descriptive purposes, men vlues were presented on untrnsformed nd undjusted vribles. Results were expressed s men SE. We used Person correltion coefficient to evlute the correltion of the vribles, nd multiple regression nlysis to investigte min fctors influencing postprndil lipid response. Following identifiction of the min fctor, subjects in upper 25th percentile of the fctor were selected nd individully mtched by ge (within 2 yer difference) nd BMI (within 1 kg/m 2 difference) to subjects within the remining 75th percentile. Selected subjects were grouped for subsequent nlyses. Chi-squred tests were used to compre differences in frequencies for ctegoricl vribles. A two tiled vlue of P 0.05 ws considered sttisticlly significnt. RESULTS Bsl nthropometricl, clinicl, nd behviorl chrcteristics of the prticipnts Tble 1 shows the bsl chrcteristics of the 102 subjects initilly selected to prticipte in this study. Their men BMI ws with rnge of 18.2 to 27.6 yers. Seventy-four men reported consumption of lcoholic beverges nd 39 were current smokers. These subjects hd similr hbitul dietry ft intke, physicl ctivity, nd socioeconomic sttus Journl of Lipid Reserch Volume 44, 2003

4 TABLE 1. Generl chrcteristics of the smple of 102 nonobese helthy men Min Mx Age (yers) (21 53) BMI (kg/m 2 ) ( ) WHR ( ) Body ft (%) ( ) Blood pressure SBP (mmhg) ( ) DBP (mmhg) (55 119) Current smoking (%) 38 Tobcco consumption (0 40) (cigrettes/dy) Current drinking (%) 72 Alcohol intke (g/dy) ( ) Hypertension (%) 16 Totl energy expenditure (kcl/dy) ( ) Totl clorie intke (kcl/dy) ( ) % ft from totl clorie intke ( ) Adipose tissue res (cm 2 ) t first lumbr vertebr Subcutneous ( ) Viscerl ( ) Adipose tissue res (cm 2 ) t fourth lumbr vertebr Subcutneous ( ) Viscerl ( ) Postprndil TG re ( ) MDA (nmol/ml) ( ) 8-epi-PGF 2 (pg/mg cretinine) ( ) BMI, body mss index; DBP, distolic blood pressure; 8-epi-PGF 2, 8-epi prostglndin F 2 ; MDA, mlondildehyde; SBP, systolic blood pressure; TG, tricylglycerol; WHR, wist-hip rtio. Men SE. Reltionship of nthropometricl prmeters to postprndil lipemi nd lipid peroxidtion The verge men vlues of subcutneous nd viscerl ft res (VFAs) t L1 nd L4 vertebre, postprndil TG re, MDA, nd 8-epi-PGF 2 of the 102 subjects re shown in Tble 1. Tble 2 shows tht some nthropometricl prmeters such s body ftness nd bdominl dipose tissue distribution (WHR nd VFA t L1 nd L4 vertebre) were positively correlted with postprndil lipemi, wheres ll vribles reflecting body ftness nd dipose tissue distribution were positively ssocited with plsm TABLE 2. Person correltions of ge, body ft distribution, totl triglyceride response re, nd lipid peroxides in 102 nonobese helthy men Postprndil TG Are MDA 8-epi-PGF 2 R P R P R P Age SBP DBP BMI WHR L1VF L1SF L4VF L4SF L1VF, viscerl ft re t first lumbr vertebr; L1SF, subcutneous ft re t first lumbr vertebr; L4VF, viscerl ft re t fourth lumbr vertebr; L4SF, subcutneous ft re t fourth lumbr vertebr. LN, log trnsformed. MDA concentrtions. BMI, WHR, subcutneous ft res t L1 nd L4 levels, nd VFA t L4 showed positive correltion with urinry excretion of 8-epi-PGF 2. Postprndil lipemi showed positively significnt correltion with plsm MDA concentrtion (R 0.332, P 0.005, dt ws not shown in Tble 2). Age ws ssocited with neither postprndil lipemi nor lipid peroxides. Identifiction of min influencing fctor on postprndil lipid response Bsed on the informtion generted from the correltion nlyses between body ft mesures nd postprndil TG re, we crried out stepwise multiple regression nlysis in order to identify the most significnt nthropometricl predictor of postprndil lipid response. Mesures of body ftness, lcohol consumption, nd cigrette smoking were used s independent vribles, nd postprndil TG re ws the dependent vrible. The dt presented in Tble 3 show tht the most significnt predictor of postprndil lipemic response ws VFA t L4 level followed by totl ft re t L4 level. Comprison of postprndil lipemi, lipid peroxidtion, DNA dmge, nd endothelil cell function between the two groups ctegorized ccording to VFA t L4 level Figure 1 shows the distribution of VFA t L4 level in ll 102 nonobese helthy men. Subjects in upper 25th percentile of the min fctor (VFA t L4 level) were ctegorized s high VFA (HVFA, n 27). The lowest VFA in this group ws over 100 cm 2 (100.3 cm 2 ) nd the verge re ws cm 2. Subjects with HVFA were ge- (within 1 yer difference) nd BMI- (within 1 kg/m 2 difference) mtched to control subjects selected for hving low VFA (LVFA; 100 cm 2, n 27). The verge re of the control group ws 74 3 cm 2. Blood pressure nd body ft distribution Men within the HVFA were chrcterized by higher WHR nd blood pressure nd higher consumption of cigrettes thn those within the LVFA group (Tble 4). Among subjects within the HVFA, 38.5% hd hypertension. No differences were found in subcutneous ft re t both L1 nd L4 vertebre (Tble 4). Nonobese men with HVFA showed higher rtio of viscerl to subcutne- TABLE 3. Stepwise multiple regression for 102 nonobese helthy men using ge, blood pressure, nthropometric prmeter, ft res t different levels of body, lcohol consumption, nd cigrette smoking s independent vribles nd postprndil TG re s dependent vrible Dependent Vrible Step Independent Adjusted Vrible R 2 R 2 Postprndil TG re 1 step L4VF step L4TF L4TF, totl ft re t 4th lumbr vertebr. Ft res t different levels of body: totl, viscerl, nd subcutneous ft res t both 1st nd 4th lumbr vertebr. LN, log trnsformed. P Jng et l. Viscerl ft, postprndil lipemi, nd lipid peroxidtion 2359

5 TABLE 5. Serum lipids, hormones, nd glucose nd responses to 75 g orl glucose tolernce test in two subgroups of nonobese men mtched on the bsis of ge nd BMI but with low versus high viscerl ft re Low VFA HVFA Fig. 1. Distribution of viscerl ft re (VFA) t 4th lumbr vertebr in 102 nonobese helthy men. ous ft re nd lower muscle re of midthigh compred with those with LVFA. Serum lipid, hormone, nd orl glucose tolernce test No differences were found in HDL cholesterol nd poa-i; however, men chrcterized by HVFA showed TABLE 4. Generl chrcteristics, ft, nd muscle res in two groups of nonobese men mtched on the bsis of ge nd BMI but with low versus high viscerl ft re Low VFA HVFA Age (yers) BMI (kg/m 2 ) Wist-hip rtio c Body ft (%) Blood pressure SBP (mmhg) b DBP (mmhg) c Current drinking (%) Alcohol (g/dy) Current smoking (%) Tobcco (cigrettes/dy) b Hypertension (%) First lumbr vertebr Totl ft (cm 2 ) b Viscerl ft (cm 2 ) b Subcutneous ft (cm 2 ) Viscerl-subcutneous ft rtio Fourth lumbr vertebr Totl ft (cm 2 ) b Viscerl ft (cm 2 ) b Subcutneous ft (cm 2 ) Viscerl-subcutneous ft rtio b Clf Ft (cm 2 ) Muscle (cm 2 ) Mid thigh Ft (cm 2 ) Muscle (cm 2 ) b HVFA, high viscerl ft re (VFA 100 cm 2, n 27); VFA, viscerl ft re. Low VFA, VFA 100 cm 2, n 27. Men SE. P 0.05 compred with low VFA. b P 0.01 compred with low VFA. c P compred with low VFA. TG (mg/dl) Totl cholesterol (mg/dl) b HDL cholesterol (mg/dl) LDL cholesterol (mg/dl) c Totl/HDL cholesterol b ApoA-I (mg/dl) ApoB (mg/dl) b Testosterone (ng/ml) IGF-1 (ng/ml) c Leptin (ng/ml) Fsting level Glucose (mg/dl) d FFA ( Eq/l) c Insulin ( IU/ml) Response re Glucose (mg/dl h) FFA ( Eq/l h) c Insulin ( IU/l h) HOMA insulin resistnce Apo, polipoprotein; FFA, free ftty cid; IGF-1, insulin-like growth fctor-1; HOMA, homeostsis model ssessment. n 27. Men SE. P 0.01 compred with low VFA. b P compred with low VFA. c P 0.05 compred with low VFA. d P 0.1 compred with low VFA. higher serum concentrtions of TG, totl cholesterol, LDL cholesterol, nd pob (Tble 5). Moreover, they hd lower serum concentrtions of testosterone nd IGF-1 nd higher fsting FFA levels thn those with LVFA. Orl glucose tolernce test (OGTT) re under the curve for glucose, insulin, nd FFA were higher in nonobese men with HVFA thn in those with LVFA (Tble 5). Lipid peroxides, DNA dmge in lymphocytes, nd vsorectivity of brchil rtery Nonobese men with HVFA showed higher concentrtions of urinry excretion of PGF 2 nd plsm MDA thn those with LVFA (Fig. 2). Til moments on the DNA in lymphocytes were lso significntly higher in nonobese men with HVFA s compred with those in the LVFA group (Fig. 2). In brchil rtery dimeters t bseline to test for FMD% nd NMD%, there were no significnt differences between LVFA group nd HVFA group (for FMD%; mm vs mm, P 0.177, nd for NMD%: mm vs mm, P 0.678). Both groups hd no significnt differences in NMD% (LVFA group: %; nd LVFA group: %); however, the HVFA group hd significntly lower FMD% compred with the LVFA group (Fig. 2). Postprndil responses of TG, glucose, FFA, nd insulin Figure 3 illustrtes concentrtions of TG, glucose, FFA, nd insulin before nd fter the mixed-mel tolernce test mong subjects with HFVA nd LVFA. Subjects within the HVFA showed higher concentrtions of TG t the 0 h, 2 h, 3 h, 4 h, nd 6 h time points nd 82% greter TG AUC compred with men in the LVFA group. TG concentr Journl of Lipid Reserch Volume 44, 2003

6 Fig. 2. Lipid peroxides, deoxyribonucleic cid (DNA) dmge of lymphocytes nd flow-medited diltion of brchil rtery in two subgroups of nonobese men mtched on the bsis of ge nd body mss index (BMI) but with low versus high VFA, men SE. * P 0.05, ** P 0.01, *** P 0.01 compred with low VFA subgroup. Low VFA, VFA 100 cm 2 ; high VFA, VFA 100 cm 2 ; TM, til moment % of DNA in the til til length ( m). 8-epi-PGF 2, 8-epiprostglndin F 2 ; FMD, flow-medited diltor. tions t 6 h were bck to fsting vlues mong men with LVFA; however, mong men with HVFA, TG concentrtions mesured t the 6 h time point remined 14% higher thn fsting vlues (P 0.032). Glucose nd insulin concentrtions t the 2 h nd 3 h time points nd fsting FFA were higher in the HVFA thn in the LVFA group. Postprndil AUCs for FFA, glucose, nd insulin were significntly incresed by 28%, 14%, nd 38%, respectively (Fig. 3), s compred with LVFA group. Totl clorie intke nd totl energy expenditure There were no significnt differences between the two groups in the proportion of energy intke derived from mcronutrients nd lcohol. However, men with HVFA showed higher totl clorie intke, lower totl energy expenditure, nd lower rtio of totl energy expenditure to totl clorie intke thn those with LVFA (Tble 6). Adjusted effects of viscerl ft t L4 level on postprndil TG re, lipid peroxides, DNA dmge, nd endothelil cell function The HVFA group hd the incresed levels of postprndil TG re, MDA, 8-epi-PGF 2, nd DNA dmge, nd decresed levels of FMD compred with LVFA group (Tble 7). However, becuse the significnt differences in smoking sttus between the LVFA nd HVFA groups we crried the nlyses fter djustment for cigrette smoking. The results of these nlyses provided similr pttern to those described for undjusted results. Fig. 3. Postprndil responses of triglyceride, glucose, free ftty cid, nd insulin to high-ft mel in two subgroups of nonobese men mtched on the bsis of ge nd BMI but with low versus high VFA men SE. * P 0.05, ** P compred with low VFA subgroup. Low VFA, VFA 100 cm 2 ; high VFA, VFA 100 cm 2. DISCUSSION Our dt show tht bdominl body ft distribution, more specificlly VFA t the L4 level, n index of metbolic complictions in obese subjects (24), ws the mjor predictor of postprndil lipemi response (mesured s TG AUC) in our popultion consisting of nonobese subjects. Moreover, we demonstrted tight ssocitions between HVFA t the L4 level, peroxidtion (mesured s urinry 8-epi-PGF 2, plsm MDA, nd DNA dmge), nd glucose tolernce (mesured s glucose, insulin, nd FFA concentrtions). Most interestingly, even for subjects TABLE 6. Usul totl clorie intke nd totl energy expenditure in two subgroups of nonobese men mtched bsed on ge nd BMI but with low versus high viscerl ft re Low VFA HVFA Totl energy expenditure Totl clorie intke % protein % crbohydrte % ft % lcohol Totl energy expenditure/ totl clorie intke b Men SE. P 0.05 compred with low VFA. b P 0.01 compred with low VFA. Jng et l. Viscerl ft, postprndil lipemi, nd lipid peroxidtion 2361

7 TABLE 7. Cigrette smoking-djusted effects of VFA t 4th lumbr vertebr on lipid peroxides, deoxyribonucleic cid dmge, nd endothelil cell function in two subgroups of nonobese men mtched on the bsis of ge nd BMI but with low versus high VFA Undjusted P Adjusted P Postprndil TG re MDA epi-PGF DNA dmge FMD Fsting glucose NS Fsting FFA Fsting insulin NS NS HOMA insulin resistnce NS NS DNA, deoxyribonucleic cid; FMD, flow-medited diltion; NS, not significnt by ANCOVA. LN, log trnsformed. with norml BMI, HVFA ws ssocited with unhelthy lifestyle fctors such s higher consumption of cigrettes nd lower rtio of totl energy expenditure to totl clorie intke compred with subjects with LVFA. Metbolic bnormlities, such s high blood pressure, high lipid concentrtions, nd incresed res of glucose, FFA, nd insulin following n OGTT, in subjects with high viscerl ft re consistent with viscerl ft syndrome proposed by Ymshit et l. (24). As viscerl dipose tissue, not subcutneous dipose tissue is drined by the portl venous system nd hs direct connection with the liver, the higher lipolytic ctivity in viscerl dipocytes leds to IR by cusing FFA levels to increse in the liver (25, 26). In glucose metbolism, response res of glucose, FFA, nd insulin were higher in HVFA group compred with LVFA group; however, s HOMA IR ws not significntly different between the two groups, this is probbly due to the fct tht chnges in fsting glucose level might interfere with the consistency of fsting insulin levels when fsting insulin level is not high. Therefore, it might be difficult to show IR with only HOMA IR using only fsting levels of glucose nd insulin (27). In ddition to the ltered fsting metbolic profile, comprison of postprndil lipid nd glucose metbolism reveled tht men chrcterized by high levels of viscerl dipose tissue presented incresed responses of TG, insulin, nd glucose fter the mixed-mel test. Moreover, glucose intolernce hs been linked to oxidtion sttus or lipid peroxidtion (28). Consistent with this notion, our dt show tht higher levels of plsm MDA nd urinry excretion of 8-epi- PGF 2 in subjects with HVFA compred with those with LVFA. In ddition, we hve shown tht urinry excretion of 8-epi-PGF 2 nd plsm MDA correlte with other more commonly used diposity indexes, such s BMI nd WHR. Nevertheless, we found some specific reltions between body ft mesures nd these two mesures of lipid peroxidtion tht my result from differences in specificity nd sensitivity in ssessing lipid peroxidtion (29, 30). MDA is generl indictor of oxidtive stress rther thn specific mrker of lipid peroxidtion nd it is known to vry in response to the diet (23, 24). Dietry fctors such s the mounts of ft intkes nd lcohol even in nonobese subjects, especilly when clorie intke is in excess of body needs, my induce the ccumultion of viscerl ft (13), which is chrcterized by its high lipogenic ctivity s well s its ccelerted lipolytic ctivity (24). Oxidtive stress, including lipid peroxidtion, cn negtively influence the ntioxidnt system (28, 29) nd consequently cuse greter DNA dmge (31). Evlution of DNA dmge in lymphocytes hs been used s biologicl mrker in the detection, monitoring, nd prognosis of chronic degenertive diseses such s therosclerosis (31). Differences in the extent of DNA dmge in the norml popultion hve been reported to not only depend on ging but lso depend on eting nd smoking hbits (9, 32). In this study, the HVFA ft group hd incresed cigrette smoking, higher lipid peroxidtion, nd consequently with higher til moment of DNA compred with subjects included in the LVFA group. However, most interestingly, even fter djusting for cigrette smoking, DNA dmge nd lipid peroxides between the two groups were still significntly different. This suggests n independent ssocition between viscerl ft ccumultion nd oxidtion. Incresed oxidtive stress is lso known to cuse endothelil dysfunction by incresed vsculr superoxide nion production nd subsequent decrese in nitric oxide (NO) biovilbility through superoxide-induced NO degrdtion (12). Endothelil dysfunction hs been lso ssocited with rnge of CVD risk fctors including smoking (33), viscerl ft ccumultion independent of BMI (34) nd subcutneous ft re (35), nd fsting or postprndil hypertriglyceridemi (11). All of these risk fctors were observed in our nonobese men with HVFA. There is substntil evidence supporting correltion between viscerl ft ccumultion nd endocrine bnormlities including low concentrtions of testosterone nd IGF-1 (4, 36, 37). Generlly, serum concentrtions of testosterone hve negtive correltion with viscerl ft ccumultion in men (38 40). Becuse testosterone decreses the lipoprotein lipse ctivity of viscerl ft cells, it cn limit the ft ccumultion in viscerl region (38 40). On the other hnd, it increses the number of -drenergic receptors nd the ctivity of hormone sensitive lipse, ccelerting the lypolysis from viscerl ft (38 40). IGF-1 lso plys n importnt role in body ft quntity nd distribution by reducing bdominl VFA, nd building up muscles nd bones (34, 35). Serum concentrtions nd the ctivities of testosterone nd IGF-1 re inversely correlted with ge, smoking, nd viscerl ft (37, 39 42). Accordingly, our study shows tht subjects within the HVFA group hd lower levels of testosterone nd IGF-1 compred with LVFA group. From this experimentl design, we cnnot conclude whether these reduced levels might cuse viscerl ft ccumultion or, lterntively, viscerl ft ccumultion itself might bring reduction in testosterone nd IGF-1 levels. Serum leptin levels were not significntly different between the two groups, in greement with reports showing tht leptin levels were relted with totl or subcutneous ft ccumultions rther thn with viscerl ft ccumultion (43, 44) Journl of Lipid Reserch Volume 44, 2003

8 Finlly, these nonobese men with HVFA showed positive energy blnce nd lower levels of muscle re t midthigh compred with their low viscerl ft counterprts, consistent with previous studies showing tht body ft nd muscle distribution relte to behviorl hbits including diet, physicl ctivity, nd smoking (36, 45). In conclusion, we found significnt correltions between bdominl ft distribution, postprndil lipemi, nd lipid peroxidtion in nonobese pprently helthy men. Specificlly, our dt show tht viscerl ft ccumultion t the L4 level is the most significnt correlte of impired postprndil lipid response, lipid peroxidtion, DNA dmge of lymphocytes, nd endothelil dysfunction, ll of them implicted in crdiovsculr morbidity nd mortlity risk. The mechnisms behind the reltionship between viscerl obesity, lipid peroxidtion, DNA dmge, nd endothelil dysfunction need to be elucidted in order to implement successful therpeutic pproches. It hs been previously shown tht viscerl dipose tissue ccumultion t L4 nd L5 is positively ssocited with C-rective protein levels, suggesting tht the expnded bdominl ft depot my induce low-grde inflmmtory stte (46, 47), which in turn will stimulte lipid peroxidtion (48). This incresed lipid peroxidtion could then promote DNA dmge, which hs been shown to contribute to vsculr dysfunction (49). Moreover, vsculr dysfunction my lso result directly from viscerl obesity independently of other conventionl risk fctors (50). A most importnt finding is the fct tht ll these ssocitions were observed mong subjects who would hve been considered norml by the commonly used nthropometricl risk fctors. This is consistent with the drmtic increses in CVD rtes observed in Kore in the bsence of significnt BMI increses, supporting the greter relevnce of ft distribution. Therefore, the mjor gol of intervention studies should be the reduction of viscerl ft through physicl exercise nd hert-helthy nutrition guidelines even for those subjects who re considered t low risk bsed on their BMI vlues. Moreover, similr studies re needed on White subjects to investigte the generlity of our findings. This study ws prtly supported by the Ministry of Helth nd Welfre, Kore; Food Ingredient Division, Foods R&D Center, CJ Corp., Seoul, Kore; Grnts from NIH/NHLBI #HL54776; nd contrcts 53-K nd from the US Deprtment of Agriculture Reserch Service. REFERENCES 1. Choo, V WHO ressesses pproprite body-mss index for Asin popultions. Lncet. 360: South Koren Ministry of Helth nd Welfre Reports on Ntionl Nutrition survey; Report on Ntionl Helth nd Nutrition Survey. Seoul, South Kore: Ministry of Helth nd Welfre. 3. South Koren Ntionl Sttisticl Office Annul Report on Cuse of Deth Sttistics. Seoul, South Kore: Ntionl Sttisticl Office. 4. Kissebh, A. H., nd G. R. 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