Apolipoprotein C-III, metabolic syndrome, and risk of coronary artery disease

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1 Apolipoprotein C-III, metbolic syndrome, nd risk of coronry rtery disese Oliviero Olivieri, 1, * Antonell Bssi, Chir Strnieri, Elisbett Trbetti, Nicol Mrtinelli,* Frncesc Pizzolo,* Domenico Girelli,* Simonett Friso,* Pier Frnco Pigntti, nd Roberto Corrocher* Unit of Internl Medicine, Deprtment of Clinicl nd Experimentl Medicine,* Institute of Clinicl Chemistry, Section of Biology nd Genetics, Deprtment of Mother nd Child nd Biology Genetics, University of Veron, Veron, Itly Abstrct Apolipoprotein C-III (poc-iii) is mrker of triglyceride (TG)-rich lipoproteins, which re often incresed in metbolic syndrome (MS). The T 455C polymorphism in the insulin-responsive element of the APOC3 gene influences TG nd poc-iii levels. To evlute the contribution of poc-iii levels nd T 455C polymorphisms in the coronry rtery disese (CAD) risk of MS ptients, we studied 873 ptients, 549 with CAD nd 251 with norml coronry rteries. Ptients were clssified lso s hving or not hving MS (MS, n 270; MS-free, n 603). Lipids, insulin, polipoprotein levels, nd APOC3 T 455C genotypes were evluted. ApoC-III levels were significntly incresed in MS ptients, nd the probbility of hving MS ws correlted with incresing qurtiles of poc-iii levels. MS ptients with CAD hd significntly higher poc-iii levels thn did CAD-free MS ptients. The crriership for the 455C vrint multiplied the probbility of CAD in MS in n llele-specific wy nd ws ssocited with incresed poc-iii nd TG levels. Obesity ws less frequent in MS crriers of the 455C llele thn in MS noncrriers (21.6% vs. 34.8%, P 0.05). In conclusion, poc-iii-rich lipoprotein metbolism nd the APOC3 polymorphism hve relevnt impcts on the CAD risk of MS ptents. Olivieri, O., A. Bssi, C. Strnieri, E. Trbetti, N. Mrtinelli, F. Pizzolo, D. Girelli, S. Friso, P. F. Pigntti, nd R. Corrocher. Apolipoprotein C-III, metbolic syndrome, nd risk of coronry rtery disese. J. Lipid Res : polipoproteins poc-iii coronry dis- Supplementry key words ese genes lipids The metbolic syndrome (MS) is clinicl entity chrcterized by obesity, hypertension, hypertriglyceridemi, low serum HDL cholesterol, nd either dibetes mellitus type 2 or glucose intolernce (1). The importnce of this clinicl clustering s single pthologicl entity ws recently defined (1), lthough the underlying etiologicl fctors re s yet mostly unknown. Ptients with MS hve n enhnced propensity to develop premture rteriosclerosis nd n incresed crdiovsculr disese mortlity nd morbidity rte (2), nd they represent, depending on ge, 24% to 42% of the US generl popultion (3). Therefore, it is of prmount importnce to identify cusl nd/or ggrvting fctors of MS, prticulrly in terms of crdiovsculr disese prevention. All of the key components of the syndrome hve genetic bsis (4, 5). As consequence, n interction or multiplictive effects of polymorphisms in number of different genes my potentilly be involved in the pthogenesis (4). Gene muttions interfering with specific insulin or hormone-responsive elements in the regultory regions hve been regrded ltely with prticulr ttention (5). In ddition to the presence of hypertriglyceridemi nd low serum HDL cholesterol, MS nd/or insulin resistnce re lso chrcterized by n increse of smll LDL prticles nd triglyceride (TG)-rich lipoproteins, fetures tht lso contribute to the crdiovsculr disese risk (4 8). One of the most importnt nd relible mrkers of TGrich lipoproteins levels is polipoprotein C-III (poc-iii). ApoC-III is 79-mino-cid protein synthesized by liver nd intestine, which is n essentil constituent of circulting prticles rich in tricylglycerol, i.e., chylomicrons nd VLDLs. ApoC-III inhibits the hydrolysis of TG-rich prticles by the lipoprotein lipse nd their heptic uptke medited by poe (9, 10). Therefore, the overexpression of the APOC3 gene results in n overt hypertriglyceridemi [s reviewed in ref. (11)]. In spite of this importnt role of poc-iii in TG metbolism, reltively few dt exist in the literture regrding the reltionships between poc-iii nd hypertriglyceridemi in MS ptients (6 8). The therogenetic role of poc-iii (12 17), s well s tht of hypertriglyceridemi in MS for coronry rtery disese Mnuscript received 11 June 2003 nd in revised form 3 September Published, JLR Ppers in Press, October 16, DOI /jlr.M JLR200 1 To whom correspondence should be ddressed. e-mil: oliviero.olivieri@univr.it Copyright 2003 by the Americn Society for Biochemistry nd Moleculr Biology, Inc Journl of Lipid Reserch Volume 44, 2003 This rticle is vilble online t

2 (CAD) risk (3 6), is well recognized. However, it is still uncler whether elevted levels of TG-rich lipoproteins nd poc-iii re highly coexpressed in MS nd wht their specific contribution is to the higher risk for crdiovsculr disese in MS ptients. The APOC3 gene is trnscriptionlly downregulted by insulin levels (18), nd sequences in the promoter region with high ffinity for the nucler trnscription fctors mediting the insulin response re highly polymorphic (19). Vrints t positions 455 nd 482 hve been shown to hve reduced ffinity for the nucler trnscription fctors mediting the insulin response (20), so tht they ppered to be the first exmple of genetic polymorphism in n insulin-responsive element nd of insulin resistnce t the gene level (20). Recently, we reported tht homozygosity for the APOC3 T 455C vrint represents n independent fctor for the susceptibility to CAD risk (21). Becuse homozygosity for the 455C llele is ssocited with incresed levels of TG nd poc-iii (21), we hypothesized possible link between the 455C insulin-resistnt vrint nd the MS phenotype, with potentil dditive interction for CAD risk. To this im, in lrge cse-control study of subjects ngiogrphiclly defined s either CAD or CAD-free, we nlyzed TG nd poc-iii levels ccording to the presence of both the MS phenotype nd the 455C llele. METHODS Study popultion We studied totl of 873 unrelted dult ptients of both genders who were recruited consecutively from those referred to the Institute of Crdiovsculr Surgery or to the Crdiovsculr- Hypertension Unit of the Deprtment of Internl Medicine of the University of Veron in Itly (the Veron Hert Project). Of these ptients, 599 hd ngiogrphiclly documented, severe multivessel coronry therosclerosis nd were cndidtes for coronry rtery bypss grfting (CAD group). As control group, we considered 274 subjects with ngiogrphiclly documented norml coronry rteries (CAD-free), exmined for resons other thn possible CAD (in 90% of cses, vlvulr hert disese). The controls were required to hve neither history nor evidence of therosclerosis in other vsculr beds. Becuse the primry im of our selection ws to provide n objective nd cler-cut definition of the therosclerotic phenotype, subjects with nonsignificnt coronry stenosis ( 50%) were not included in the study. At the time of blood smpling, complete clinicl nd phrmcologicl history, including the presence or bsence of the trditionl crdiovsculr disese risk fctors, ws obtined. According to these dt, ptients were clssified s hving MS (MS group) when t lest three of the following elements were present: body mss index (BMI) 30 kg/m 2, cliniclly documented history of hypertension or blood pressure 140/90, fsting glucose 110 mg/dl, plsm TG 150 mg/dl, nd HDL cholesterol 40 (50 for femles) mg/dl. All the remining ptients (including those with only one or two of the bove described clinicl fetures) were considered to be MS-free subjects. The study ws pproved by our institutionl review bords. Either written or orl informed consent ws obtined from ll ptients. Biochemicl nlysis Smples of venous blood were drwn from ech subject in the free-living stte fter n overnight fst. Serum lipids nd the other routine biochemicl prmeters were determined s previously described (21). Insulin ws mesured by n immunometric sndwich ssy (Immulite 2000 Insulin) from Dignostic Products Corportion, Los Angeles, CA; intr- nd interssy vrition coefficients of the method were 5%. To obtin n estimte of insulin resistnce, we pplied the homeostsis model ssessment (HOMA) of insulin resistnce using the following formul: HOMA fsting insulin ( IU/ml) fsting glucose (mmol/l)/ 22.5 (22). ApoA-I, pob, nd poe were mesured by commercilly vilble nephelometric immunossys; ntiser, clibrtors nd BNII nephelometer were from Dde Behring, Mrburg, Germny. Intr-sssy vrition coefficient ws clculted on 10 control replictes nd interssy on duplictes over 10 dys. Imprecision ws within mnufcturer specifictions, i.e., the intrssy vrition coefficients were 2.1%, 1.6%, nd 1.98%, nd interssy vrition coefficients were 3.2%, 2.36%, nd 3.98% for poa-i, pob, nd poe, respectively. ApoC-III ws mesured by fully utomted turbidimetric immunossy. The regents were obtined from Wko Pure Chemicl Industries (Osk, Jpn), nd the procedure recommended by the mnufcturer ws implemented on n RXL Dimension Anlyzer (Dde Interntionl Inc., Newrk, DE). Imprecision ws ssessed on three pools of control ser with low, medium, nd high concentrtions of poc-iii; intr-ssy vrition coefficients were 1.84%, 2.02%, nd 1.98%, nd interssy vrition coefficients were 4.4%, 3.4%, nd 2.29% for low, medium, nd high concentrtion, respectively. In subgroup of ptients (CAD, n 80; CAD-free, n 36), poc-iii ws mesured in whole serum s well s heprin-mn superntnts nd heprin- Mn precipittes. In this wy, poc-iii ssocited with HDL or with LDL VLDL frctions ws seprtely quntified. Genotype nlysis Genomic DNA ws extrcted from whole blood smples by the phenol-chloroform procedure, nd ll subjects were genotyped for the APOC3 T 455C polymorphism s previously described (21). Sttisticl nlysis All computtions were performed using the SPSS 10.0 sttisticl pckge (SPSS Inc., Chicgo, IL). Distributions of continuous vribles were expressed s mens SD. Logrithmic trnsformtion ws performed for skewed vribles, i.e., for poc-iii nd TG, nd the sttisticl differences concerning these prmeters were lso computed on the corresponding log-trnsformed vlues, lthough, for the ske of simplicity nd clerness, nontrnsformed dt re reported in the Results. Sttisticl significnce for differences in quntittive vribles ws ssessed by Student s unpired t-test, nd it ws lso tested by one-wy ANOVA djusted for ge nd/or sex (Generl Liner Model procedure). Qulittive dt were nlyzed by the 2 test. Correltion between log-trnsformed totl poc-iii (mesured in whole serum) nd log-trnsformed poc-iii ssocited with HDL or ssocited with LDL-VLDL ws evluted by Person coefficient. The T 455C llele nd genotype frequencies were compred, by 2 nlysis, with the vlues predicted on the bsis of the Hrdy-Weinberg equilibrium. Lipid vribles were compred mong ptients with different genotypes by ANOVA, using the Tukey procedure for post hoc multivrite comprison of the mens. Odds rtio (OR) nd 95% confidence intervl (95% CI) for CAD or MS were clculted by logistic regression nlysis. In prticulr, to ssess the extent to which APOC3 genotypes nd MS were ssocited with CAD, the popultion ws strtified in six ptient groups (TT, TC, nd CC genotypes, with or without MS) Olivieri et l. ApoC-III nd metbolic syndrome 2375

3 nd OR with 95% CI ws estimted by logistic-regression nlysis. To provide seprte ORs for ech genotype, dummy vribles were used, considering MS-free TT genotype s the reference group. Adjustment for the risk fctors conventionlly not ssocited with MS (ge, gender, smoking sttus, nd totl cholesterol) ws performed by including these covrites in second set of multivrite logistic regression models. A regression model for forml interction between MS nd the T 455C genotype ws lso built to estimte the CAD risk proportion ssocited with the MS genotype term. RESULTS The clinicl chrcteristics nd the T 455C genotype frequencies of CAD nd CAD-free ptients re summrized in Tble 1. CAD ptients hd more conventionl risk fctors nd significntly higher poc-iii levels thn did CADfree ptients (Tble 1). Age (OR for CAD 1.033; 95% CI, ), mle gender (OR 1.96; 95% CI, ), LDL cholesterol (OR 1.456; 95% CI, ), nd smoking (OR 2.62; 95% CI, ) were the min predictors of CAD risk. Interestingly, the T 455C polymorphism nd MS were lso significntly ssocited with CAD risk. Both the 455C llele (0.403 vs ; OR for CAD 1.304; 95% CI, ) nd genotype frequency (OR for CAD 1.96; 95% CI, ) were significntly higher in CAD thn in CAD-free individuls (Tble 1). The presence of MS ws the strongest predictor of risk: TABLE 1. Prmeters Chrcteristics of ptients with or without coronry rtery disese CAD Ptients (n 599) CAD-Free (n 274) P Age (yers) Mle sex (%) BMI (kg/height 2 ) b Cholesterol b Totl (mmol/l) LDL (mmol/l) HDL (mmol/l) TGs (mmol/l) b ApoA-I (g/l) b ApoB (g/l) b ApoC-III (mg/dl) b ApoE (mg/dl) b NS Insulin ( IU/ml) b NS Uric cid (mmol/l) b NS Current smoking (%) Hypertension (%) Dibetes (%) MS ptients (%) C llele frequency (95% CI) T 455C genotypes 455 TT (%) 222 (37.1) 115 (42) 455 TC (%) 271 (45.2) 131 (47.8) 455 CC (%) 106 (17.7) 28 (10.2) 0.02 ApoA-I, polipoprotein A-I; BMI, body mss index; TG, triglyceride; MS, metbolic syndrome; CAD, coronry rtery disese; 95% CI, 95% confidence intervl. Sttisticl significnce for differences ws tested by Student s unpired t-test or by 2 test when pproprite. Vlues re mens SD. b Age- nd sex-djusted vlues. totl of 270 subjects presented the fetures of MS, nd they were more represented in the CAD group thn in the CAD-free group (38.1% vs. 15.3%; OR for CAD 3.39; 95% CI, ). The clinicl chrcteristics nd the T 455C genotype frequencies of ptients ccording to MS nd MS-free groups re reported in Tble 2. The two groups were mtched for ge nd totl nd LDL cholesterol levels, but they differed in severl other spects, both relted nd unrelted to the clssic MS elements (see Tble 2), nd 40% of MS ptients presented overt insulin resistnce (HOMA in the upper qurtile of the distribution). MS ptients were chrcterized by incresed levels of poc-iii nd poe (Tble 2). In prticulr, 74% of MS ptients hd poc-iii vlues higher thn the medin distribution vlue of the MS-free popultion ( 10.2 mg/dl). To evlute whether the increse in poc-iii is common feture in MS ptients, we then computed the risk for MS ssocited with poc-iii levels divided in qurtiles distribution of the study popultion s whole. As shown in Fig. 1, OR for MS ws directly relted with poc-iii levels divided in qurtiles; this reltionship reched sttisticl significnce for poc-iii levels mg/dl (vlues corresponding to the third or fourth qurtile), nd ws confirmed even fter djustment for ge, sex, totl cholesterol, poa-i, pob, poe, TG, nd the other MS elements. In the ptient smples nlyzed by mens of heprin- Mn centrifugtion, totl poc-iii ws strongly correlted with non-hdl poc-iii concentrtion (R 0.93; P ) nd much more wekly correlted with HDL-ssocited poc-iii (R 0.38; P 0.001). Prmeters TABLE 2. Chrcteristics of ptients with or without MS MS Ptients (n 270) MS-Free (n 603) P Age (yers) NS Mle sex (%) BMI (kg/height 2 ) b Glucose (mmol/l) b Cholesterol b Totl (mmol/l) NS LDL (mmol/l) NS HDL (mmol/l) TG (mmol/l) b ApoA-I (g/l) b ApoB (g/l) b ApoC-III (mg/dl) b ApoE (mg/dl) b Insulin ( IU/ml) b Uric cid (mmol/l) b HOMA, upper qurtile (%) Current smoking (%) Hypertension (%) C llele frequency NS (95% CI) ( ) ( ) T 455C genotypes 455 TT (%) 99 (36.7) 238 (39.5) 455 TC (%) 130 (48.1) 272 (45.1) 455 CC (%) 41 (15.2) 93 (15.4) NS HOMA, homeostsis model ssessment. Sttisticl significnce for differences ws tested by Student s unpired t-test or by 2 test when pproprite. P vlue ws considered significnt when Vlues re mens SD. b Sex-djusted vlues Journl of Lipid Reserch Volume 44, 2003

4 Fig. 1. Odds rtio for metbolic syndrome (MS) in reltion to the percentiles distribution of polipoprotein C-III in the whole popultion. 95% CI, 95% confidence intervl. There ws no difference in T 455C genotype distribution between MS nd MS-free ptients, nd no increse of MS risk ws ssocited with the mutnt llele (Tble 2). Differences between MS ptients with or without CAD were lso nlyzed (Tble 3). The two groups of MS ptients were well mtched for most prmeters, including poa-i nd HDL cholesterol levels, but differed for lipid vribles such s totl cholesterol, TG, poc-iii, pob, nd poe levels (Tble 3). When the T 455C genotype distribution ws renlyzed, seprting the popultion into different subgroups bsed on both MS/MS-free nd CAD/CAD-free sttus, n evident symmetry ws observed (Tble 4): very high proportion of MS ptients, heterozygous or homozygous for the 455C vrint, were ffected by CAD (87% nd 92.7%, respectively), s compred with 58.1% nd 73.1% in the corresponding genotype groups without MS (Tble 4). On the bsis of this observtion, in order to ssess the TABLE 3. Chrcteristics of MS ptients, seprted into two groups ccording to the ngiogrphic evidence of CAD Prmeters CAD Ptients (n 228) CAD-Free (n 42) P Age (yers) NS Mle sex (%) NS BMI (kg/height 2 ) NS Cholesterol Totl (mmol/l) LDL (mmol/l) HDL (mmol/l) NS TGs (mmol/l) ApoA-I (g/l) NS ApoB (g/l) ApoC-III (mg/dl) ApoE (mg/dl) HOMA, upper qurtile (%) NS Uric cid (mmol/l) NS Current smoking (%) NS Hypertension (%) NS Dibetes (%) NS Sttisticl significnce for differences ws tested by Student s unpired t-test or by 2 test when pproprite. P vlue ws considered significnt when Vlues re mens SD. extent to which APOC3 genotypes nd MS could interct in determining CAD risk, we estimted the OR for CAD fter strtifying the whole popultion in six groups (TT, TC, nd CC genotypes, with or without MS) by logistic regression nlysis. The results obtined using MS-free ptients with TT genotype s the reference group re detiled in Fig. 2. Homozygosity for the 455C llele ws by itself ssocited with CAD risk in both MS nd MS-free conditions. Similrly, the presence of MS ppered to be significntly ssocited with n incresed OR for CAD, regrdless of the genotype (OR for 455TT MS group 2.24; 95% CI, ). The most striking finding, however, ws the strong grded increse of risk ssocited with the crriership of the 455C llele in cses of coexisting MS phenotype, with one mutnt llele ssocited with fctor 2 of multipliction of risk (Fig. 2). Adjustment for the vribles not included in the MS definition (ge, sex, totl cholesterol, nd smoking) did not chnge the results (for the MS-free 455CC subgroup, OR 1.69; for the MS 455TT subgroup, OR 2.17; for the MS 455TC subgroup, OR 3.93; for the MS 455CC subgroup, OR 8.5). To confirm the interction between MS nd poc3 genotype, forml model including the interction term between these vribles ws lso crried out, nd results re reported in Tble 5. To evlute whether the higher risk for CAD relted to the presence of the APOC3 455C vrint ws to be scribed to the bnorml lipid metbolism in subject crriers of the muttion, we performed further nlyses. After exclusion of individuls treted with sttins or fibrtes t the time of blood smpling (n 310, most of them CAD ptients), lipid nd polipoprotein profiles in the different genotype groups with or without MS were, therefore, compred. A totl of 563 ptients were then nlyzed, nd the results re reported in Tble 6. As expected, MS ptients hd less poa-i nd HDL cholesterol nd higher TG nd poc-iii levels thn did MS-free subjects, but homozygous mutnts for T 455C lso hd more pronounced increse in TG nd poc-iii in comprison with the other MS genotype groups (Tble 6). The difference between groups for poc-iii did not, however, rech sttisticl significnce, probbly due to the reltively smll smple size. In spite of cler TG- nd poc-iii-rising trend for the 455CC group, the genotype effects considered seprtely in the subgroup of MS-free subjects were sttisticlly null; in MS subjects, on the contrry, 455C homozygosity ws ssocited with significntly higher TG but not with higher poc-iii levels (Tble 6). The further reduction of smple size, however, limited the vlue of both of these results. Finlly, we exmined the reltive expression of MS elements in subjects either crrying or not crrying one mutnt llele. There ws no significnt difference in the degree of hyperglycemi, hypertension, low HDL cholesterolemi, or hypertriglyceridemi between the two groups, but obesity ws significntly less frequent in 455C crriers s compred with the noncrrier group (21.6% vs. 34.8%; P 0.05). The groups were well mtched for ll other clinicl nd biochemicl chrcteristics, but 455C crri- Olivieri et l. ApoC-III nd metbolic syndrome 2377

5 TABLE 4. Apo C-III genotype distribution in ptients with or without MS, with or without CAD Apo CIII Genotype (No MS) ApoC III Genotype (With MS) Dignosis 455TT 455TC 455CC 455TT 455TC 455CC Totl Ptients CAD-free ptients Within DG (%) Within genotype (%) CAD ptients Within DG (%) Within genotype (%) Totl ptients ers hd BMIs sttisticlly lower thn did ptients with the 455TT genotype ( kg/m 2 vs kg/m 2 ; P 0.05). DISCUSSION The results of the present study suggest tht incresed poc-iii levels re common feture of the MS phenotype. ApoC-III levels were, indeed, significntly incresed in the mjority of MS ptients, nd the probbility of hving MS ws directly correlted with poc-iii levels divided into qurtiles (Fig. 1). Moreover, relevnt interction ble to modulte CAD risk ws observed between MS phenotype nd the crriership of n APOC3 vrint, predisposing to n insulin resistnt, bnorml poc-iii production. Genetic nd/or cquired mechnism(s) leding to the development of MS my, directly or indirectly, imply n incresed poc-iii synthesis, due either to gene ctivtion nd/or to stbiliztion of its trnscript, or to decresed ctbolism of the protein. The former mechnism my resonbly be invoked in the cse of crriership of the 455C genetic vrint. It hs been shown tht the most frequent promoter vrint, 455T, is ssocited with 40 50% insulin-medited downregultion of APOC3 gene expression (20). In contrst, the less common 455C vrint seems ssocited with the complete loss of the insulinmedited suppression of APOC3 gene trnscription (20). Bsed on these findings, 455C crriership should led, in vivo, to n incresed synthesis of poc-iii in proportion rnging from 25% to 50%, depending on the presence of the vrint in heterozygosis nd homozygosis, even in the presence of n effective cell insulin ction. A recent study reported for the first time the reference limits for poc-iii in lrge, helthy, French Cucsin popultion, in which the level corresponding to the 50th percentile for middle-ged men ws 10 mg/dl (23). Considering the French vlues s reference, our MS Itlin Cucsins with either the 455TC or 455CC genotype hd poc-iii levels 29% nd 53% higher, respectively. Ptients with the sme genotypes but without MS showed, however, increments of poc-iii by 7% nd 11.3%, respectively. Using the poc-iii men vlues observed for 455TT Fig. 2. Odds rtio for coronry rtery disese in different genotype groups of ptients with or without MS Journl of Lipid Reserch Volume 44, 2003

6 TABLE 5. Interction model between T 455C genotype nd MS on CAD risk Covrites Coefficient Significnce OR (95% CI) T 455C genotype TC ( ) 455CC ( ) MS ( ) Interction term TC MS ( ) 455CC MS ( ) OR, odds rtio. By logistic regression. MS-free subjects in our popultion s reference (10.6 mg/dl, see Tble 4), we observed tht percent increses of poc-iii for MS ptients were substntilly similr to those reported in the French study (22% nd 44% of increment for heterozygous nd homozygous, respectively). There were none or only mrginl differences (6.6%) in comprison with the corresponding genotype groups of MS-free ptients. Therefore, the comprison of the dt predicted by in vitro gene expression studies with those obtined in our humn study suggests tht the poc-iii-rising effect inherent in the genetic vrint cnnot be expressed in the bsence of MS nd becomes detectble only with the coexistence of MS. The opposite sitution does not necessrily occur. In fct, incresed poc-iii levels in MS do not require the presence of the 455C vrint. Indeed, poc-iii ws incresed lso in noncrriers with MS (Tble 6), nd no difference in distribution of T 455C genotypes ws observed between MS nd MS-free ptients (Tble 2). This observtion excludes n etiologic role for the gene vrint in MS nd implies tht other independent poc- III-rising mechnisms, such s reduced protein ctbolic rte, lso hve to be ctivted in MS (8). In this context, it should be interesting to verify the role of other recently discovered TG-rising genetic vrints on the sme poa3/a4/a5 genes cluster (24 26). Similrly, we cnnot exclude the possibility tht the 455C muttion is in linkge disequilibrium with these new vrints, resulting in being merely mrker for some of them. Our findings indicte, however, tht the interction between the insulin-resistnt T 455C gene polymorphism nd MS seems to ply synergic role in the expression of MS-ssocited lipid bnormlity nd in its impct on CAD risk. In our study, MS ptients ffected or not by CAD differed exclusively in lipid metbolism prmeters, i.e., totl nd LDL cholesterol, pob, poc-iii, nd poe levels (Tble 3), suggesting primry role for lipid bnormlities in CAD risk ssocited with MS. Furthermore, no differences were observed in poa-i nd HDL cholesterol levels between the two groups. Incresed pob nd cholesterol in circulting lipid prticles is well-known feture in CAD ptients, but the notion of incresed poc-iii nd poe levels in MS ptients with CAD is not generlly ccepted. Sprse informtion concerning the different MS elements, such s obesity (7, 8, 27) or type II dibetes (28, 29), exist, generlly confirming the reltion between hypertriglyceridemi nd increse of poc-iii nd poe, but there re no specific studies reporting on MS s unique complex. To the best of our knowledge, there re no studies evluting the CAD risk of MS ptients in reltion to the extent of poc-iii increse. Severl reports hve independently confirmed the role of poc-iii in incresing CAD risk without distinguishing between ptients with or without MS (12 17). It is possible tht more ccurte informtion on CAD risk could be obtined by evlution of non-hdl poc-iii frction (17), nd the lck of vilbility of these prmeters for nlysis in our study is certinly limittion of the present work. However, in subgroup of ptients, totl poc-iii concentrtion ws much more strongly correlted with non-hdl frction (R 0.93) thn with HDL poc-iii (R 0.38), suggesting tht the informtive power of the totl concentrtion of the polipoprotein should be similr to tht given by the frction not ssocited with HDL. In our popultion, MS ws ssocited per se with n odds rtio for CAD of 3.39 (95% CI, ). Consistent with our previous report (21), 455C homozygosity ws lso ssocited with CAD per se, regrdless of coexisting MS (see Fig. 2, 455CC no MS group). The new nd TABLE 6. Lipid nd polipoprotein profile in the different genotype groups of ptients with or without MS, nd free of hypolipidemic therpy (n 563) Without MS With MS APOC3 Genotype 455TT (n 155) 455TC (n 182) 455CC 455TT (n 54) P b (n 63) 455TC (n 86) 455CC (n 23) P b LDL cholesterol (mmol/l) NS NS HDL cholesterol (mmol/l) NS c c c NS TGs (mmol/l) NS c c c,d 0.05 ApoA-I (g/l) NS c c c NS ApoB (g/l) NS e e NS ApoC-III (mg/dl) NS c c c NS ApoE (mg/dl) NS NS Comprison between ll six groups of ptients: b Comprison between genotype groups in MS or MS-free ptients, respectively (by ANOVA, post hoc Tukey test). c Significntly different (P 0.05) from every group of ptients without MS (by ANOVA, post hoc Tukey test). d Significntly different (P 0.05) from every other group of ptients (by ANOVA, post hoc Tukey test). e Significntly different (P 0.05) from 455TC group of ptients without MS (by ANOVA, post hoc Tukey test). Olivieri et l. ApoC-III nd metbolic syndrome 2379

7 interesting finding ws tht grded, strong interction in determining CAD risk emerged when both MS nd crriership of the 455C gene vrint coexist in the sme individul (Fig. 2, Tble 5). This result is importnt for t lest two resons: i) it strongly supports the preeminent role of poc-iii-rich lipoproteins in incresing CAD risk in MS ptients, becuse the mthemticl reltion ( fctor 2) we observed between the grded ORs for CAD risk nd 455C heterozygosity or homozygosity is difficult to explin without ccepting the poc-iii-rising effect of the gene vrint on n llelic bsis; nd ii) strong interction ws lso demonstrted in the cses of heterozygosis for the 455C llele, thus extending the potentil impct of the gene vrint in terms of generl popultion risk. Becuse in the generl popultion, 50 70% of individuls re 455C crriers (30 33), the risk deriving from the interction of this polymorphism with cquired or lifestyle fctors fvoring the development of MS is potentilly very relevnt. Among ll fctors, one of the most importnt is probbly obesity (34) or, more generlly, condition of incresed clorie vilbility derived from excess limentry intke. Its role hs been prticulrly stressed in the cse of the ssocition of hyperlipidemi with MS nd insulin resistnce, in which liver bundnce of free ftty cids flux stimultes the ssembly nd secretion of TG-rich lipoproteins (35). Recently, obese men with insulin resistnce (men BMI, kg/m 2 ) were demonstrted to hve higher plsm poc-iii nd TG-rich lipoprotein levels nd lower estimted frctionl ctbolic rte of these prticles (8). Both incresed synthesis nd reduced ctbolic rte of poc-iii rich prticles seem, therefore, to be involved in hyperlipidemi observed in MS ptients, lthough the reltive contribution of ech mechnism remins to be specified. In the cse of our MS ptients, obese individuls (BMI 30 kg/m 2 ) were significntly less numerous in 455C crriers thn in noncrriers (21.6% vs. 34.8%), nd crriers hd significntly lower BMIs thn did noncrriers. There ws no pprent reson for this difference (prticulrly considering tht it is the result of post hoc nlysis) unless cusl effect is ttributed to 455C crriership. If reduced ctbolic rte of poc-iiirich lipoproteins is the previling mechnism leding to hyperlipidemi in obesity (8), then obese individuls should be preferentilly found in the group of 455C noncrriers rther thn in the 455C crriers. Another resonble interprettion is tht in 455C crriers, reltively smller BMI increse nd liver ftty cid flux re required to trigger the cscde of metbolic events peculir to MS, i.e., n incresed synthesis of TG nd poc-iii. 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