Rocio Vera

Manuel Sánchez

Milagros Galisteo

Inmaculada Concepcion Villar

Rosario Jimenez

Antonio Zarzuelo

Francisco Pérez-Vizcaíno

Juan Duarte
email: jmduarte@ugr.es

Inmaculada Concepcion Villar

Chronic administration of genistein improves endothelial dysfunction in spontaneously hypertensive rats: involvement of eNOS, caveolin and calmodulin expression and NADPH oxidase activity

Keywords: Genistein, SHR, phytoestrogen, hypertension, eNOS, caveolin, calmodulin, superoxide

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Introduction

Isoflavones (e.g. genistein and daidzein), constitute the main class of phytoestrogens. They are found in abundance in soybeans and their derivative foods.

There is evidence that the beneficial cardiovascular effects of consumption of soy are mostly due to its isoflavone fraction [START_REF] Cassidy | Biological effects of a diet of soy protein rich in isoflavones on the menstrual cycle of premenopausal women[END_REF][START_REF] Wong | Cholesterol-lowering effect of soy protein in normocholesterolemic and hypercholesterolemic men[END_REF][START_REF] Nestel | Soy isoflavones improve systemic arterial compliance but not plasma lipids in menopausal and perimenopausal women[END_REF]. The cardioprotective ability of these plant products has been attributed, at least in part, to their ability to decrease plasma cholesterol, to lower blood pressure and to improve arterial compliance [START_REF] Cassidy | Biological effects of a diet of soy protein rich in isoflavones on the menstrual cycle of premenopausal women[END_REF][START_REF] Wong | Cholesterol-lowering effect of soy protein in normocholesterolemic and hypercholesterolemic men[END_REF][START_REF] Nestel | Soy isoflavones improve systemic arterial compliance but not plasma lipids in menopausal and perimenopausal women[END_REF]. Genistein (4',5,7-trihydroxy-isoflavone) in vitro relaxes rat arteries through a NO-dependent mechanism [START_REF] Mishra | Endothelium-dependent relaxation of rat aorta and main pulmonary artery by the phytoestrogens genistein and daidzein[END_REF] and enhances the dilator response to acetylcholine in coronary arteries from atherosclerotic female macaques [START_REF] Honore | Soy isoflavones enhance coronary vascular reactivity in atherosclerotic female macaques[END_REF] and in aortae from both ovariectomized rats [START_REF] Squadrito | Genistein supplementation and estrogen replacement therapy improve endothelial dysfunction induced by ovariectomy in rats[END_REF] and male spontaneously hypertensive rats (SHR) [START_REF] Vera | Soy isoflavones improve endothelial function in spontaneously hypertensive rats in an estrogen-independent manner: role of nitric-oxide synthase, superoxide, and cyclooxygenase metabolites[END_REF]. Moreover, the phytoestrogen daidzein enhanced eNOS activity increasing calmodulin, its endogenous activator, and decreasing caveolin-1 in normotensive male rats [START_REF] Sobey | Effect of short-term phytoestrogen treatment in male rats on nitric oxide-mediated responses of carotid and cerebral arteries: comparison with 17beta-estradiol[END_REF]. In vivo, genistein has also been reported to improve endothelial function in endotoxemic rats [START_REF] Bermejo | In vivo vascular effects of genistein on a rat model of septic shock induced by lipopolysaccharide[END_REF] and SHR [START_REF] Kitayama | Chronic administration of a tyrosine kinase inhibitor restores endothelial functional and morphological changes of the basilar artery during chronic hypertension[END_REF]. In addition, genistein or soy supplements have been proven to be effective in protecting endothelial function in men and healthy postmenopausal women [START_REF] Squadrito | The effect of the phytoestrogen genistein on plasma nitric oxide concentrations, endothelin-1 levels and endothelium dependent vasodilation in postmenopausal women[END_REF][START_REF] Colacurci | Effects of soy isoflavones on endothelial function in healthy postmenopausal women[END_REF]. However, the mechanisms involved in its beneficial effect on endothelial function are unclear.

Genistein binds to estrogen receptors (ERs) α and β, but it shows greater affinity for the ERβ [START_REF] Makela | Differentiation between vasculoprotective and uterotrophic effects of ligands with different binding affinities to estrogen receptors alpha and beta[END_REF]. Besides its activity on ERs, the effects of genistein have been also attributed to its inhibitory properties on a broad range of tyrosine kinases [START_REF] Bermejo | In vivo vascular effects of genistein on a rat model of septic shock induced by lipopolysaccharide[END_REF][START_REF] Kitayama | Chronic administration of a tyrosine kinase inhibitor restores endothelial functional and morphological changes of the basilar artery during chronic hypertension[END_REF][START_REF] Duarte | Effect of tyrosine kinase and tyrosine phosphatase inhibitors on aortic contraction and induction of nitric oxide synthase[END_REF][START_REF] Nevala | Genistein treatment reduces arterial contractions by inhibiting tyrosine kinases in ovariectomized hypertensive rats[END_REF], to its peroxisome proliferator-activated receptor γ agonist activity [START_REF] Dang | Peroxisome proliferator-activated receptor gamma (PPARgamma) as a molecular target for the soy phytoestrogen genistein[END_REF] or its antioxidant activity [START_REF] Xu | Genistein inhibits expressions of NADPH oxidase p22phox and angiotensin II type 1 receptor in aortic endothelial cells from stroke-prone spontaneously hypertensive rats[END_REF]. We have recently demonstrated that, in vitro, genistein and its analog daizdein acutely improved endothelial function in arteries from SHR, an effect unrelated to its estrogenic properties [START_REF] Vera | Soy isoflavones improve endothelial function in spontaneously hypertensive rats in an estrogen-independent manner: role of nitric-oxide synthase, superoxide, and cyclooxygenase metabolites[END_REF].

Therefore, the aim of the present study was to further characterize the mechanisms of the in vivo effects of genistein on endothelial function, and particularly, the role of ER. Thus, we analyzed the effects of chronic oral genistein administration on endothelial function and the potential mechanisms involved, focusing on the NO pathway: eNOS activity, the expression of eNOS and its regulatory proteins (calmodulin-1 and caveolin-1). The role of estrogenic mechanisms was studied using a pure α and β ER antagonist. We selected male hypertensive animals to analyze the female sex-independent effects of dietary genistein. Genistein was used at doses which resulted in serum genistein concentrations similar to those in humans eating a traditional Asian diet high in soy [START_REF] Adlercreutz | Plasma concentrations of phyto-oestrogens in Japanese men[END_REF][START_REF] Fritz | Dietary genistein: perinatal mammary cancer prevention, bioavailability and toxicity testing in the rat[END_REF].

Methods

Animals and experimental groups

Male Wistar Kyoto (WKY) and SHR aged 23 weeks were obtained from Harlan Laboratories (Barcelona, Spain). All rats were maintained five per cage at a constant temperature (24 ± 1ºC), with a 12-hour dark/light cycle and on standard rat chow without soy derivatives (AIN 76). The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No, 85-23, revised 1996).

Experiment I. Analysis of the influence of genistein on blood pressure and endothelial function in SHR.

Rats were divided into the following groups (n = 10 in each group), WKY-vehicle (methylcellulose 1% w/v); SHR-vehicle; WKY-Genistein (10 mg/kg, per day, by gavage); SHR-Genistein. Drugs were given for 5 weeks. Body weight was measured every week. The genistein treatment was stopped 2 days before the end of the experiment in order to study the long-term effects of genistein without the involvement of the effects of acute administration.

Experiment II. Analysis of involvement of estrogenic mechanisms in the effects of genistein in SHR. Rats were divided into the following groups (n = 7 in each group), SHR-vehicle; SHR-Genistein (10 mg/kg, per day, by gavage) and SHR-Genisteinfaslodex (ICI 182,780, kindly provided from AstraZeneca, Cheshire, England) (2.5 mg/kg, per day, s.c.). Drugs were given for 5 weeks. Body weight was measured every week. The genistein treatment was stopped 2 days before the end of the experiment.

Blood pressure measurements and plasma sexual hormone levels

Systolic blood pressure (SBP) was measured 18-20 hours after administration of the drugs in conscious, prewarmed, restrained rats by tail-cuff plethysmography [START_REF] Duarte | Antihypertensive effects of the flavonoid quercetin in spontaneously hypertensive rats[END_REF]. At least seven determinations were made in every session and the mean of the lowest three values within 5 mmHg was taken as the SBP level. At the end of experiment I, direct blood pressure and heart rate were measured in four conscious rats per group. For this purpose, the rats were anaesthetized with 2.5 ml/kg equitensin (500 mL contain 43% (w/v) chloral hydrate in 81 mL ethanol; 4.86 mg nembutal; 198 mL propylene glycol; 10.63 g MgSO4; distilled water) (i.p.). A polyethylene catheter (PE-50) containing 100 U heparin in isotonic, sterile, NaCl solution was inserted into the right femoral artery for intra-arterial blood pressure and heart rate measurement in conscious rats. The catheter was tunnelled subcutaneously, exteriorized through the skin on the dorsal side of the neck and protected with a silver spring. Rats were allowed to recover for 24 h and, after connecting the catheter to a transducer and a two-channel recorder (TRA-021 and Letigraph 2000, respectively; Letica SA, Barcelona, Spain), blood pressure and heart rate were continuously recorded for 60 min. Plasma testosterone and estradiol were measured by electrochemiluminescence immunoassay (Roche Diagnostics, Mannheim, Germany).

Vascular reactivity studies

Descending thoracic aortic rings ( and 5% CO 2 . Rings were stretched to 2 g of resting tension by means of two L-shaped stainless-steel wires inserted into the lumen and attached to the chamber and to an isometric force-displacement transducer (Letigraph 2000), respectively, as previously described [START_REF] Duarte | Antihypertensive effects of the flavonoid quercetin in spontaneously hypertensive rats[END_REF]. The concentration-relaxation response curves to acetylcholine (10 -8 -10 -4 M) were performed in rings pre-contracted by 10 -6 M phenylephrine. The concentrationrelaxation response curves to nitroprusside (10 -9 -10 -6 M) were performed in the dark in rings pre-contracted by 10 -6 M phenylephrine. Endothelium-dependent contractions to acetylcholine were tested in rings which were initially stimulated with 80 mM KCl. After washing in Krebs solution and incubation for 30 min with L-NAME (10 -4 M), acetylcholine was added in a cumulative fashion (10 -8 -10 -4 M). In these experiments, the contractile responses to acetylcholine were expressed as a percentage of the response to KCl. Copyright 2006 The Biochemical Society phenylmethylsulphonyl fluoride (1 mg/ml) and leupeptin (10 μg/ml). Samples homogenates were incubated at 37ºC for 30 min in TRIS buffer (50 mM, pH 7.4) in the presence of the NADPH (1 mM), L-valine (10 mM) and a mixture of unlabelled and L-[ 3 H]arginine (10 μM, 1 μCi/ml). Reactions were terminated by the addition of 20 mM HEPES (1 ml, pH 5.5) containing 1mM EGTA and 1 mM EDTA. L-[ 3 H]citrulline was separated from arginine by adding 1.5 ml of a 1:1 suspension of cation exchange resin (Dowex AG50 W-X8, Sigma) in water. The radioactivity was measured in the supernatants by liquid scintillation counting. The activity of the Ca 2+ -dependent NOS was determined from the difference between the L-[ 3 H]citrulline produced from samples containing Ca 2+ and samples without Ca 2+ and with 1 mM EGTA. Results were expressed as picomoles of L-citruline per milligram of protein per 30 minutes.

Measurement of vascular NOS activity

Western blotting analysis

Frozen aortic rings were homogenized with a glass potter. eNOS, caveolin-1 and calmodulin-1 protein expression was measured in the supernatant of aortic homogenates.

Proteins were quantified using the Bradford method. Western blottings were performed with 40 μg protein per lane for eNOS, caveolin-1 and calmodulin-1. Sodium dodecyl sulphate (SDS)-polyacrilamide (8% for eNOS or 15 % for the rest of proteins) electrophoresis was performed in a mini-gel system (Bio-Rad Laboratories, Hercules, California, USA). The proteins were transferred to polyvinylidene difluoride membranes (PVDF) and incubated overnight with primary monoclonal mouse anti-eNOS (1:2500 dilution, Transduction Laboratories, San Diego, California, USA), monoclonal mouse anticaveolin-1 (1:600 dilution, Transduction Laboratories), or polyclonal goat anti-calmodulin-1 (1:1000 dilution, SantaCruz Biotechnology, Santa Cruz, California, USA). The membranes were then washed five times for 10 min in Tris-buffered saline-0.1 % Tween 20 (TBST) and incubated with the corresponding secondary peroxidase conjugated goat anti-mouse or rabbit anti-goat antibodies (1:2000, Sigma-Aldrich, Madrid, Spain). All incubations were performed at room temperature for 2 hours. After washing the membranes, antibody binding was detected by an ECL system (Amersham Pharmacia Biotech, Amersham, UK). Films were scanned and densitometric analysis was performed on the scanned images using Scion Image-Release Beta 4.02 software (http://www.scioncorp.com). As an additional control, samples were re-probed for expression of α-skeletal actin. Protein abundance/α-actin ratio was performed and results were presented as comparisons to this ratio in aorta from WKY-vehicle in experiment I or from SHR-vehicle in experiment II on the same gels, expressed as 100%.

Detection of vascular superoxide (O

Statistical analysis

Results are expressed as means ± s.e. means of measurements. Two way analysis of variance (ANOVA) was used to compare responses of control (vehicle-treated) animals with those of treated ones, and control SHR with control WKY rats. In experiment II, comparisons were made by one way ANOVA, with post-hoc multiple comparisons using Dunnet test. In all cases a P < 0.05 was considered statistically significant. Concentration-response curves were fitted to the logistic equation:

E = E max / (1 + 10 -k * ([drug]-pD2) )
, where E max is the maximal effect, k is a factor which represents the slope of the curve, and pD 2 is the drug concentration exhibiting 50% of the E max expressed as negative log molar.

Results

Blood pressure, heart rate and plasma sexual hormones levels

Long-term genistein administration reduced tail SBP in SHR and this effect reached statistical significance after 17-18 days of treatment while no changes were observed in WKY rats (Figure 1A, Experiment I). Tail SBP values were confirmed by direct mean arterial pressure measurements in conscious animals at the end of the experimental period (Figure 1A). Heart rate was increased in control SHR as compared to control WKY, but genistein did not significantly modify this parameter in both SHR and WKY rats (Figure 1A). The pure ER antagonist faslodex was unable to modify the antihypertensive effect induced by chronic oral genistein treatment (Figure 1B, experiment II).

Plasma testosterone and estradiol levels were similar in control WKY (

Ex vivo vascular reactivity

In aortae from WKY and SHR treated with genistein, no differences were observed in the vasoconstrictor responses induced by noradrenaline or in the endothelium-independent vasodilator responses to the NO donor sodium nitroprusside as compared with their respective control WKY and SHR groups (Table 1). Aortae from vehicle-treated SHR showed significant reduced endothelium-dependent vasodilator responses to acetylcholine in arteries stimulated by phenylephrine as compared to aortae from control WKY (E max = 32.5 ± 5.2 % vs 71.9 ± 4.1 %, respectively, P<0.05) (Figure 2A). In addition, in the presence of L-NAME (10 -4 M), aortae from SHR also showed greater endothelium-dependent vasoconstrictor responses to acetylcholine than their normotensive WKY counterparts (48.7 ± 3.9 % vs 15.6 ± 4.8 % of the response to 80 mM KCl, respectively). Acetylcholine was unable to induce a contraction in denuded vessels from WKY or SHR. Genistein produced a significant increase in the relaxation induced by acetylcholine only in SHR (Figure 2A), without changes in the endotheliumdependent vasoconstriction to acetylcholine in both SHR and WKY (45.7 ± 8.1 % and 9.6 ± 1.7 % of the response to 80 mM KCl, respectively). Figure 2B shows that faslodex was unable to modify the improvement in endothelial function induced by genistein in SHR (Experiment II).

Effects of genistein on vascular eNOS activity and vascular protein expression of eNOS and its protein regulators

Compared with the WKY control group, untreated SHR exhibited a significant reduction of aortic eNOS activity (Figure 3A). Genistein treatment increased eNOS activity in SHR but not in WKY (Figure 3A, experiment I) and this increase was not modified by faslodex (Figure 3B, experiment II). Paradoxically, protein expression of eNOS in the aorta was much higher in SHR than in the corresponding WKY group and genistein treatment had no effect on either group (Figure 4A). Thus, we examined the changes in expression of caveolin-1, the allosteric negative regulator of eNOS, and calmodulin-1, which positively regulates eNOS activity. The expression of caveolin-1 and calmodulin-1 was markedly lower in aortae from SHR control compared to WKY (Figure 4B, and 4C). After treatment with genistein, caveolin-1 protein expression was unchanged in both WKY and SHR (Figure 4B), while calmodulin-1 protein was increased only in SHR (Figure 4C). In SHR arteries without endothelium the expression of calmodulin-1 was lower than in intact arteries, but there were no differences among groups (not shown), indicating that the changes in calmodulin-1 occurred in the endothelium and not in the smooth muscle. In experiment II, aortic rings from faslodex plus genistein showed up-regulated eNOS protein as compared to genistein group (Figure 5A). The expression of caveolin-1 and calmodulin-1 in the faslodex plus genistein group was similar to the genistein group (Figure 5B and 5C), i.e. faslodex did not prevent the increase in calmodulin-1 expression induced by genistein.

Effects of genistein on vascular production of O 2 -

No significant differences were found in basal vascular production of O 2 -within groups as measured by the chemiluminescence of lucigenin (ranking between 0.16-0.26 nmol/min/mg tissue). Aortic production of O 2 -stimulated by NADPH was significantly higher in SHR than WKY control rats (Figure 6). Preincubation of some aortic rings with the inhibitor of NADPH oxidase DPI almost suppressed the O 2 -production induced by NADPH. Chronic treatment with genistein induced a significant decrease of NADPHstimulated O 2 -production only in SHR, being without effects in WKY rats (Figure 6).

Discussion

Long-term oral treatment of male hypertensive rats with the soy isoflavone genistein restores the impaired aortic endothelium-dependent vasodilatation and reduces blood pressure, without effect on normotensive WKY rats. The major new findings of the present study were: (1) The genistein-induced improvement on endothelial function in SHR was associated with enhanced eNOS activity, increased expression of its modulator calmodulin-1 and reduced vascular production of the NO scavenger O 2 -.

Changes in endothelial function, blood pressure and protein expression induced by genistein seems to be unrelated to ER activation because co-administration of the pure α and β ER antagonist faslodex did not modify the effects of genistein.

In vitro, soybean-derived phytoestrogens genistein and daidzein and the mammalian estrogen 17β-estradiol, improved the endothelial function in isolated arteries from male hypertensive animals, restoring the impaired relaxant response to acetylcholine [START_REF] Vera | Soy isoflavones improve endothelial function in spontaneously hypertensive rats in an estrogen-independent manner: role of nitric-oxide synthase, superoxide, and cyclooxygenase metabolites[END_REF]. However, there are multiple factors (including pharmacokinetics, homeostasis and feedback mechanisms, hormonal control and changes in gene expression) which are not retained in in vitro studies and may influence the long term in vivo effects. In the present in vivo study and in agreement with Kitayama et al. [START_REF] Kitayama | Chronic administration of a tyrosine kinase inhibitor restores endothelial functional and morphological changes of the basilar artery during chronic hypertension[END_REF], we showed that chronic oral genistein treatment increased the endothelium-dependent vasodilatation to acetylcholine in male SHR but not in WKY rats. In the rat aorta, endothelium-dependent vasodilatation relies almost entirely on the endothelial release of NO and the subsequent activation of soluble guanylate cyclase [START_REF] Nagao | Heterogeneous distribution of endothelium-dependent relaxations resistant to NG-nitro-L-arginine in rats[END_REF]. The endothelium-independent vasodilation induced by the soluble guanylate cyclase activator nitroprusside was similar in SHR and WKY and unaffected by genistein, indicating that endothelial dysfunction and its improvement by genistein are due to changes in endothelium-derived NO availability rather than downstream effects on vascular smooth muscle. Additionally, apparent changes in endothelium-dependent vasodilation may also occur as a consequence of the opposing effects of the release of endothelium-derived vasoconstrictors [START_REF] Yang | A diffusible substance(s) mediates endothelium-dependent contractions in the aorta of SHR[END_REF]. In fact, consistent with previous reports [START_REF] Yang | A diffusible substance(s) mediates endothelium-dependent contractions in the aorta of SHR[END_REF], SHR showed increased endothelium-dependent vasoconstriction induced by acetylcholine in arteries treated with the NO synthase inhibitor L-NAME. This response was abolished by both endothelial denudation (present results) and incubation with the thromboxane-endoperoxide receptor antagonist SQ-29,548 [START_REF] Yang | A diffusible substance(s) mediates endothelium-dependent contractions in the aorta of SHR[END_REF], indicating the involvement of endothelial release of vasoconstrictor prostanoids. However, chronic genistein treatment did not modify the endothelium-dependent vasoconstriction induced by acetylcholine suggesting that the protective effect of genistein on endothelial function is unrelated to changes in endothelium-dependent vasoconstriction. Taken together, these data strongly suggest that genistein restores endothelial function in SHR by increasing NO availability.

The pathological reduction of NO synthesis and its restoration by drugs may be caused by several factors such as changes in the expression of eNOS, postransductional modification of the enzyme, interactions with its endogenous modulators or suboptimal concentrations of the substrate L-arginine or the cofactor tetrahydrobiopterin (BH4) [START_REF] Huang | Endothelial nitric oxide synthase and endothelial dysfunction[END_REF]. As previously reported [START_REF] Sanchez | Quercetin downregulates NADPH oxidase, increases eNOS activity and prevents endothelial dysfunction in spontaneously hypertensive rats[END_REF][START_REF] Chou | Alterations of nitric oxide synthase expression with aging and hypertension in rats[END_REF], the basal aortic eNOS activity of SHR was significantly lower than that of WKY. Interestingly, genistein enhanced eNOS activity in SHR, explaining, at least partly, the improvement of endothelium-dependent relaxation. In addition, in SHR we also found an increase in eNOS expression and a decrease in caveolin-1 expression, a negative eNOS modulator, which may even be viewed as a compensatory mechanism to maintain the production of bioactive NO in the face of increased oxidant stress [START_REF] Piech | Differential regulation of nitric oxide synthases and their allosteric regulators in heart and vessels of hypertensive rats[END_REF]. The enhancement of aortic eNOS activity in SHR by genistein may be related, at least in part, to the higher endothelial calmodulin (positive regulator of eNOS) abundance despite the unchanged eNOS and caveolin-1 protein expression, as compared to vehicle treated SHR. Cell stimulation with Ca 2+mobilizing agonists promotes calmodulin binding to eNOS which disrupts the inactive heteromeric complex formed between eNOS and caveolin, rendering the active form of the enzyme. Thus, calmodulin overexpression induced by genistein may lead to hyperactivation of eNOS and elevations of NO production in acetylcholine-vasodilation in intact aorta.

In addition, reduced endothelial NO levels may result from an increase in the oxidative inactivation of NO by O 2 -. Endothelial dysfunction in SHR has been associated in many reports with an excess of O 2 -generation [START_REF] Cai | Endothelial dysfunction in cardiovascular diseases: the role of oxidant stress[END_REF]. In our study, NADPHstimulated O 2 -production was increased in SHR aortic rings compared to WKY. We also found that genistein only reduced NADPH-stimulated aortic O 2 -production in rings from SHR but not from WKY. These results suggest that genistein specifically reduce the NADPH oxidase overactivity in SHR. Several subunits of the NADPH oxidase complex have been shown to be overexpressed in SHR [START_REF] Sanchez | Quercetin downregulates NADPH oxidase, increases eNOS activity and prevents endothelial dysfunction in spontaneously hypertensive rats[END_REF][START_REF] Cai | Endothelial dysfunction in cardiovascular diseases: the role of oxidant stress[END_REF]. We speculate that Copyright 2006 The Biochemical Society chronic genistein might also restore this protein expression as previously observed with the flavonoid quercetin [START_REF] Sanchez | Quercetin downregulates NADPH oxidase, increases eNOS activity and prevents endothelial dysfunction in spontaneously hypertensive rats[END_REF]. Therefore, an increased NO bioavailability induced by chronic genistein, seems to be due to both an increased NO synthesis and a reduced NADPH-dependent O 2 -production. The increase in NO bioavailability together with the reduced O 2 -production may account for the reduction of blood pressure induced by genistein in SHR.

Genistein is a ligand for estrogen receptor ERα and ERβ, showing greater affinity for the ERβ [START_REF] Makela | Differentiation between vasculoprotective and uterotrophic effects of ligands with different binding affinities to estrogen receptors alpha and beta[END_REF][START_REF] Duarte | Effect of tyrosine kinase and tyrosine phosphatase inhibitors on aortic contraction and induction of nitric oxide synthase[END_REF]. Squadrito et al. [START_REF] Squadrito | Genistein supplementation and estrogen replacement therapy improve endothelial dysfunction induced by ovariectomy in rats[END_REF] found that genistein exerted similar protective effects in endothelial function to 17β-estradiol in ovariectomized rats. The role of the interaction of genistein with ER was analyzed using the pure ER antagonist, faslodex. This drug was unable to alter the changes induced by genistein in blood pressure, endothelial function, eNOS activity and calmodulin aortic protein expression in SHR, suggesting that these effects are unrelated to ER stimulation. Potential alternative mechanisms include inhibition of tyrosine kinases, a large family of protein kinases involved in many signalling transduction pathways regulating gene expression.

These results agree with our previous in vitro experiments showing that genisteininduced improvement in endothelial function in SHR in vitro [START_REF] Vera | Soy isoflavones improve endothelial function in spontaneously hypertensive rats in an estrogen-independent manner: role of nitric-oxide synthase, superoxide, and cyclooxygenase metabolites[END_REF] were unaffected by antagonism of ERα and ERβ. In contrast, faslodex has been reported to prevent genistein-induced increases in eNOS mRNA in ovariectomized rats [START_REF] Wang | Genistein supplementation stimulates the oxytocin system in the aorta of ovariectomized rats[END_REF] despite that they used a lower dose of faslodex (1.5 mg/kg/day s.c) during a shorter period of time (10 days). Faslodex is classically considered the gold-standard pure ERα and ERβ antagonist [START_REF] Osborne | Fulvestrant: an estrogen receptor antagonist with a novel mechanism of action[END_REF]. However, faslodex may fail to inhibit certain acute ERβ-mediated effects [START_REF] Wade | Estrogen receptor (ER)alpha and ERbeta exhibit unique pharmacologic properties when coupled to activation of the mitogen-activated protein kinase pathway[END_REF]. Therefore, it cannot be categorically excluded the involvement of ERβ in the effects of genistein in our experiments. Surprisingly, in our study, faslodex plus genistein increased eNOS protein expression as compared to genistein. The significance of this effect is unclear because it resulted in no changes in eNOS activity. It might be interpreted in terms of the inhibitory effects of ERα and ERβ on the activity of other transcriptional factor involved in eNOS protein expression, such as peroxisome proliferator-activated receptor (PPAR)-γ. In fact, activation of ERα and ERβ downregulates PPAR-γ transcriptional activity [START_REF] Dang | Peroxisome proliferator-activated receptor gamma (PPARgamma) as a molecular target for the soy phytoestrogen genistein[END_REF]. So, it might be speculated that faslodex could facilitate the PPAR-γ agonist activity of genistein [START_REF] Dang | Peroxisome proliferator-activated receptor gamma (PPARgamma) as a molecular target for the soy phytoestrogen genistein[END_REF], leading to an increase in eNOS expression.

The major conclusion to be drawn from this study is that oral treatment with the isoflavone genistein resulted in an enhanced vascular eNOS activity in SHR associated with an increased calmodulin-1 protein expression and a reduced vascular O 2 production. Faslodex did not prevent these effects suggesting that ERs are not involved.

These findings provide novel evidence to understand how genistein improves vascular endothelial function in hypertension and further support the beneficial effects of this dietary factor in cardiovascular diseases associated with endothelial dysfunction. Copyright 2006 The Biochemical Society Copyright 2006 The Biochemical Society

  NOS activity in aortic homogenates was determined by monitoring the conversion of L-[ 3 H]arginine to L-[ 3 H]citrulline. Briefly, isolated aortic rings were homogenized, on ice, in a buffer containing 10 mM N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid (HEPES) (pH 7.4), sucrose (0.32 M), EDTA (100 μM), dithiothreitol (1 mM), Clinical Science Immediate Publication. Published on 28 Sep 2006 as manuscript CS20060185

  intact aortic segments was quantified by lucigenin-enhanced chemiluminescence, as previously described by Ohara et al.[START_REF] Ohara | Hypercholesterolemia increases endothelial superoxide anion production[END_REF]. Aortic rings from all experimental groups were incubated for 30 minutes at 37 ºC in HEPES-containing physiological salt solution (pH 7.4) of the following composition (in mM): NaCl 119, HEPES 20, KCl 4.6, MgSO 4 1, Na 2 HPO 4 0.15, KH 2 PO 4 0.4, NaHCO 3 1, CaCl 2 1.2 and glucose 5.5. Aortic production of O 2 -was stimulated by addition of NADPH (100 μM). Rings were then placed in tubes containing physiological salt solution with NADPH, and lucigenin was injected automatically at a final concentration of 5 μM. Changes in O 2 -release were determined by measuring luminescence over 200 s in a scintillation counter (Lumat LB 9507, Berthold, Germany) in 5-s intervals. In a set of experiments of control SHR and WKY, vessel segments were incubated for 30 min with the inhibitor of NADPH oxidase diphenyleneiodonium (DPI, 10 μM). Vessels were then dried, and dry Clinical Science Immediate Publication. Published on 28 Sep 2006 as manuscript CS20060185 Copyright 2006 The Biochemical Society weight was determined. O 2 -release is expressed as nmol/min/mg dry aortic tissue, using combinations of xanthine and xanthine oxidase as an internal standard.

Figure 1 .

 1 Figure 1. Effects of genistein on arterial blood pressure and heart rate. Panel A (experiment I)

Figure 2 .

 2 Figure 2. Endothelium-dependent relaxations induced by acetylcholine (Ach). Panel A

Figure 3 .

 3 Figure 3. eNOS activity measured by the conversion of L-[ 3 H]arginine to L-[ 3 H]citrulline in

Figure 4 .

 4 Figure 4. Western blots of eNOS (A), caveolin-1 (B), and calmodulin-1 (C) protein

Figure 5 .

 5 Figure 5. Western blots of eNOS (A), caveolin-1 (B), and calmodulin-1 (C) protein

Figure 6 .

 6 Figure 6. NADPH (100 μM)-stimulated O 2 -production in arteries from SHR vehicle (n = 10),

  Figure 1

Clinical Science Immediate Publication. Published on 28 Sep 2006 as manuscript CS20060185

Clinical Science Immediate Publication. Published on 28 Sep 2006 as manuscript CS20060185

Table 1 . Parameters of the concentration-response curves to endothelium-independent vasoactive factors

 1

		Noradrenaline	SNP
		(10 -9 -10 -6 M)	(10 -9 -10 -6 M)
		-log EC 50 E max (g)	-log IC 50 E max (%)
	Experiment I
	WKY-vehicle (n = 10)	8.27 ± 0.10 2.27 ± 0.27 7.36 ± 0.10 98.4 ± 1.0
	WKY-genistein (n = 10)	8.20 ± 0.05 2.66 ± 0.33 7.58 ± 0.17 97.8 ± 2.4
	SHR-vehicle (n = 10)	7.94 ± 0.17 1.92 ± 0.26 7.36 ± 0.15 97.0 ± 2.1
	SHR-genistein (n = 10)	7.79 ± 0.10 1.92 ± 0.21 7.46 ± 0.22 96.4 ± 4.8
	Experiment II
	SHR-vehicle (n = 7)	7.83 ± 0.06 1.30 ± 0.18 7.94 ± 0.10 99.1 ± 1.0
	SHR-genistein (n = 7)	7.89 ± 0.09 1.45 ± 0.17 7.94 ± 0.08 96.8 ± 2.4
	SHR-genistein-faslodex (n = 7) 7.80 ± 0.06 1.31 ± 0.08 7.86 ± 0.15 97.5 ± 2.7
	Values are expressed as mean ± SEM. E max (maximal effect) for noradrenaline is expressed as g of contraction
	and for sodium nitroprusside (SNP) as a percentage of relaxation of the pre-contraction with phenylephrine.
	Potency (-log EC 50 ) is the drug concentration exhibiting 50 % of the E max expressed as negative log molar. Potency (-
	log IC 50 ) is the drug concentration relaxing 50 % of the pre-contraction with phenylephrine expressed as negative
	log molar.

Clinical Science Immediate Publication. Published on 28 Sep 2006 as manuscript CS20060185

Clinical Science Immediate Publication. Published on 28 Sep 2006 as manuscript CS20060185 Copyright 2006 The Biochemical Society

Support: This work was supported by Grants from Comisión Interministerial de Ciencia y Tecnología SAF 2001-2953 and AGL2004-06685-C04/ALI (Acción Movilizadora de Alimentos Funcionales).


Umberto Morbiducci

Giacomo Di Benedetto

Alexandra Kautzky-Willer

Giovanni Pacini

PhD Andrea Tura
email: tura@isib.cnr.it

Improved usability of the Minimal Model of insulin sensitivity based on automated approach and Genetic Algorithms for parameter estimation

Keywords: Insulin Action, Glucose Effectiveness, Evolutionary Algorithms, Former Gestational Diabetes, Mathematical Modeling

Minimal Model analysis of glucose and insulin data from an intravenous glucose tolerance test is widely used to estimate insulin sensitivity. However, the use of the model often requires intervention by a trained operator, and some problems in the estimation of the model parameters can occur. In this study a new method for Minimal Model analysis, GAMMOD, was developed based on Genetic Algorithms for the estimation of the model parameters. Such algorithm does not require fixing initial values for the parameters (that may lead to unreliable estimates). Our method also implements an automated weighting scheme not requiring manual intervention of the operator, thus improving the usability of the model. We studied a group of 170 women with a history of previous gestational diabetes. Results obtained by GAMMOD were compared to those obtained by a traditional gradient-based algorithm for minimal model analysis, MINMOD. Insulin sensitivity by GAMMOD was 3.86±0.19 •10 -4 µU ml -1 min -1 vs. 4.33±0.20 by MINMOD; glucose effectiveness was 0.0236±0.0005 min -1 vs. 0.0229±0.0005. The difference in the estimations by the two methods are within the precision expected for such kind of metabolic parameters and are of no clinical relevance. Moreover, both the coefficient of variations of the estimated parameters and the error of fit are generally lower in GAMMOD, despite the fact that it does not require manual intervention.

In conclusion the GAMMOD approach for parameter estimation in the Minimal Model provides reliable estimation of the model parameters and improves the usability of the model, thus facilitating its further diffusion and application in a clinical contest.

Introduction

The Minimal Model is widely used to analyze glucose and insulin data from intravenous glucose tolerance tests (IVGTT) for estimating insulin sensitivity in both clinical and epidemiological studies [START_REF] Bergman | Quantitative estimation of insulin sensitivity[END_REF][START_REF] Avogaro | The hot but not the cold minimal model allows precise assessment of insulin sensitivity in NIDDM subjects[END_REF][START_REF] Mykkanen | The relation of proinsulin and insulin to insulin sensitivity and acute insulin response in subjects with newly diagnosed type II diabetes: the Insulin Resistance Atherosclerosis Study[END_REF][START_REF] Saad | A comparison between the minimal model and the glucose clamp in the assessment of insulin sensitivity across the spectrum of glucose tolerance[END_REF]. The great part of the strategies for the estimation of the model parameters resort to traditional nonlinear least squares algorithms. However, these algorithms might lead to estimates that are wrong or at least characterized by wide uncertainty [START_REF] Saad | A comparison between the minimal model and the glucose clamp in the assessment of insulin sensitivity across the spectrum of glucose tolerance[END_REF][START_REF] Pillonetto | Minimal model S I =0 problem in NIDDM subjects: nonzero bayesian estimates with credible confidence intervals[END_REF][START_REF] Pillonetto | Numerical non-identifiability regions of the minimal model of glucose kinetics: superiority of bayesian estimation[END_REF]. As assessed by Godsland and Walton [START_REF] Godsland | Maximizing the success rate of minimal model insulin sensitivity measurement in humans: the importance of basal glucose levels[END_REF], the choice of the strategy for the parameter estimation is a fundamental issue, because it may lead to failure in the convergence to the solution of the equations. Moreover, different initial values of the parameters to be estimated could produce different solutions [START_REF] Morbiducci | Genetic algorithms for parameter estimation in mathematical modeling of glucose metabolism[END_REF].

The search of a robust method for parameter estimation in Minimal Model analysis led us to select Genetic Algorithms (GAs), which are theoretically and empirically proven to provide a robust search in a complex space [START_REF] Lin | Hybrid simplex genetic algorithm for blind equalization using RBF networks[END_REF][START_REF] De | Analysis of the behaviour of a class of genetic adaptive systems[END_REF][START_REF] Goldberg | Genetic Algorithms in Search, Optimization and Machine Learning[END_REF]. GAs are random search algorithms for nonlinear problems based on the rules of natural selection. They do not require fixing an initial value for the model parameters to be estimated.

Another limitation of the Minimal Model is that it often requires the intervention of an experienced operator to produce reliable results [START_REF] Hayes | A strategy to enhance the usability of the minimal model as used with the intravenous glucose tolerance test in diabetes research and clinical management[END_REF]. A strategy to limit operator actions would be of great value to improve the usability of the model.

In this study we have developed a new method to estimate the parameters of the Minimal Model in humans, based on Genetic Algorithm approach, GAMMOD. Also, an automated strategy in the model application was developed to reduce the manual intervention by the operator. Our results by GAMMOD were compared to those obtained by the classic MINMOD, the traditional algorithm for Minimal Model analysis [START_REF] Pacini | MINMOD: a computer program to calculate insulin sensitivity and pancreatic responsively from the frequently sampled intravenous glucose tolerance test[END_REF].

G S ) t ( G )) t ( X S ( dt ) t ( dG ⋅ + ⋅ + - = (1) 0 G G(0)= [ ) I ) t ( I ( S ) t ( X p dt ) t ( dX b I 2 - ⋅ - ⋅ - = ] (2) 0 ) 0 ( X = G(t) [mg/dl]
is the plasma glucose concentration at time t; I(t) [µU/ml] is the plasma insulin concentration at time t; G b and I b are basal glucose and insulin values; X(t) [min -1 ] is insulin action at time t; S G is the glucose effectiveness [min -1 ], i.e. the glucose disappearance rate from plasma without any change in dynamic insulin concentration; S I represents insulin sensitivity [µU ml -1 min - 1 ], which is defined as the ability of insulin to enhance glucose disappearance and inhibit glucose production; G 0 is the value of plasma glucose concentration extrapolated at time zero after glucose injection; p 2 is a parameter related to the dynamics of insulin action. The parameters to be estimated are given by the vector P = [S G , p 2 , S I , G 0 ] T .

Parameter estimation through traditional algorithm: MINMOD

We estimated the unknown parameter vector P by using the Minimal Model computer program MINMOD [START_REF] Pacini | MINMOD: a computer program to calculate insulin sensitivity and pancreatic responsively from the frequently sampled intravenous glucose tolerance test[END_REF], which resorts to traditional weighted nonlinear least squares approach, using particularly the Levenberg-Marquardt algorithm for the minimization procedure. MINMOD also uses a weighting scheme of the glucose data where the weights are equal to the inverse of the variance of the measurement errors. A zero-weighting scheme is used for the early part of the glucose time course, where glucose concentration data may be unreliable since some mixing effects may still be present. It is in charge of the operator to define for each subject's data the time interval where the zero-weighting scheme should be applied. In the MINMOD algorithm the choice of the Parameter estimation through automated approach based on Genetic Algorithms: GAMMOD A weighting scheme not requiring operator intervention was implemented to be applied to the early part of the plasma glucose curve, where some mixing effects can be present. In particular, such scheme was applied over the samples collected in the first 10 minutes of the FSIGT:

(i) the first three samples, considering also the pre-injection sample, are automatically zeroweighted;

(ii) the following samples can be zero-weighted according to the following conditions:

a) if the sample G(t i ) is lower than G(t i+1 ), then G(t i ) is zero-weighted; b) if (G(t i )-G(t i+1 )) > 3•((G(t i+1 )-G(t i+2 )), then G(t i ) is zero-weighted.
Condition b) is based on the hypothesis that plasma glucose should decline rapidly and smoothly in the first minutes after the glucose injection; thus, samples that in this period remain higher or in general much different from the neighbouring samples can be considered unreliable; therefore, they are not weighted in the analysis. However, the number "3" in the condition b) is not based on theoretical reasons but only on our experience in FSIGT analysis.

No weighting scheme was adopted on glucose samples not matching conditions a) and b), and on samples following the 10 minutes interval.

We then applied GAs for the minimization procedure needed to estimate the unknown parameter vector P. Genetic Algorithms, firstly introduced by Holland in 1975 [START_REF] Holland | Adaptation in Natural and Artificial Systems[END_REF], are the best-known class of evolutionary algorithms. The evolutionary techniques mimic the principles of natural selection of living entities. They combine elements of direct and stochastic search, and exhibit some advantages over other search methods: in particular, the need of a smaller amount of a priori knowledge and, especially, fewer assumptions about the characteristics of the search space. In fact, they do not require fixing initial values for the estimates, and they usually converge to the global minimum of the error function [START_REF] Michalewicz | How to Solve It: Modern Heuristics[END_REF]. In the last years the level of interest in GAs application to engineering problems has grown considerably [START_REF] Goldberg | Genetic Algorithms in Search, Optimization and Machine Learning[END_REF]. Moreover, they have been shown to outperform alternative search techniques on difficult problems involving discontinuous, noisy, high dimensional and multimodal objective functions [START_REF] Joines | On the Use of Non-Stationary Penalty Functions to Solve Constrained Optimization Problems with Genetic Algorithms[END_REF].

In our study, each possible solution of the minimization procedure was a "chromosome", which was composed by 4 "genes", e.g., the four components of the P vector. The limits of the parameters to be estimated, defining the borders for the field of existence of the solution, were set according to our experience as follows: S G from 0.001 to 0.200 min -1 ; p 2 from 0 to 1 min -1 ; S I from 0.01 to 15 • 10 -4 µU ml -1 min -1 ; G 0 from 100 to 600 mg dl -1 .

Overcoming the problem of the possible dependence of the initial values of the parameters [START_REF] Morbiducci | Genetic algorithms for parameter estimation in mathematical modeling of glucose metabolism[END_REF], the Genetic Algorithm was started with 150 randomly generated chromosomes, and all of them were evaluated. To do that, an appropriate cost function (the so-called "fitness function") was built up.

We considered the standard sum of squares function:

2 N 1 i i m i e i m ) (t G ) (t G ) (t G ∑ = ⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛ - = Ψ (3)
G m (t i ) is the glucose experimental sample at time t i , and G e (t i ) is the corresponding model estimation, obtained by numerical solution and integration of the equation system 1) and 2); N is the number of measured samples.

The estimation in each subject's data of the unknown parameter vector P through the minimization of function Ψ was considered the objective of the GA algorithm. However, as GAs seek to maximize the fitness function, to accomplish the transformation of the minimization problem into a maximization problem the error can be simply subtracted from a large positive constant, as suggested by Karr et al. [START_REF] Karr | Least median squares curve fitting using a genetic algorithm[END_REF]. Thus, the goal was to maximize the function ff defined as ff = D -Ψ , where D is a large positive constant parameter.

For each chromosome (i.e. possible solution) the fitness function was evaluated, so that those providing high values of the fitness function were selected. After the evaluation step, two genetic operators were applied for creating new chromosomes from those selected before:

(i) the "cross-over" operator randomly picked up two parents P1, P2 and performed a sort of interpolation between them by generating a random number Ω (Ω ∈ (0, 1)), and creating two children C1 and C2 as follows:

C1 = P1 • Ω + P2 • (1-Ω); C2 = P1 • (1-Ω) + P2 • Ω. (4)
(ii) the "non-uniform mutation" operator randomly picked up one parent and changed one of its genes (i.e., one of the model parameter) on the basis of a non-uniform probability distribution. This operator searched the space of the solution uniformly at the beginning, and locally as the current generation approached the maximum number of generations.

The new population was formed by the children (derived from the application of the genetic operators) and from some high performance individuals of the old population that were reintroduced by a proper selection function, thus avoiding problems of stagnation [START_REF] Joines | On the Use of Non-Stationary Penalty Functions to Solve Constrained Optimization Problems with Genetic Algorithms[END_REF][START_REF] Karr | Least median squares curve fitting using a genetic algorithm[END_REF][START_REF] Brooks | Automatic correlation and calibration of noisy sensor readings using elite genetic algorithms[END_REF]. The previous evaluation step was repeated on the newly created population, and all the procedure was iterated. To avoid convergence to a local minimum, and to find the minimum number of iterations required for proper convergence (i.e., the number of generations necessary to the desired evolution), a sensitivity study was carried out (data not shown), as in Morbiducci et al. [START_REF] Morbiducci | Genetic algorithms for parameter estimation in mathematical modeling of glucose metabolism[END_REF]: it was found that a number of 10000 generations was sufficient to obtain an accurate estimation of P.

The numerical implementation of the whole method was performed with a proper own code using Matlab ® (The MathWorks, Inc.) Copyright 2006 The Biochemical Society

Data analysis

Statistical comparisons between the model parameters estimated by MINMOD and GAMMOD on each individual were performed by applying Deming regression analysis [START_REF] Combleet | Incorrect least-squares regression coefficients in method-comparison analysis[END_REF].

Data and results are reported as mean±SE unless otherwise designated. Precision of estimates was expressed as the coefficient of variation CV (i.e., fractional SD), calculated from the variance values in the main diagonal of the inverse of the Fisher's information matrix.

Results

Mean glucose and insulin data used in our study and GAMMOD-derived fit are reported in Figure 1. Table 1 summarizes the values of the four model parameters S G , p 2 , S I , G 0 estimated by GAMMOD approach. The corresponding coefficients of variation are also shown. Only in one case we found extremely high coefficient of variations for p 2 and S I (1398 and 138%, respectively). In the same case, however, MINMOD performed even worse (coefficient of variation for S I equal to 373%).

To compare the two parameter estimation approaches, we focused on the two most relevant parameters, i.e., insulin sensitivity, S I , and glucose effectiveness, S G (Table 2): GAMMOD estimated S I values were slightly lower than the MINMOD values, and similar results were found for S G . However, GAMMOD provided coefficients of variations lower than in MINMOD; in particular that of S I was almost half with GAMMOD than with MINMOD.

To investigate the relationship between the estimation of S I and S G by the two methods, we applied the Deming regression analysis. High values of R were found for both S I and S G : R=0.94 and R=0.86, respectively (see Figure 2 and Figure 3). Deming regression also showed that in S I the slope of the regression line was virtually coincident with the unit line (slope: 0.971; 95% confidence interval: from 0.913 to 1.029); therefore, there was no proportional difference in S I estimates by the two methods. However, since the intercept of the regression line was different from zero (intercept:

-0.345; 95% confidence interval: from -0.563 to -0.127), the two methods slightly differ by a constant amount in S I estimates. As regards S G , Deming regression showed that there was neither proportional (slope: 1.041; 95% confidence interval: from 0.903 to 1.179) nor constant difference (intercept: -0.0003; 95% confidence interval: from -0.0034 to 0.0028) by the two methods. Bland-Altman test provided similar findings for both S I and S G (not shown).

In terms of goodness of fit, GAMMOD performed better than MINMOD: the sum of square error averaged over all the study subjects was 608±37 (mg dl -1 ) 2 in MINMOD, and only 290±9 (mg dl -1 ) 2 in GAMMOD.

Discussion

The Minimal Model is the reference method to estimate insulin sensitivity and glucose effectiveness from IVGTT data. However, the reliability and accuracy of the parameter estimation may strongly depend upon the used numerical approach. Different strategies may yield extremely different parameter estimates and serious numerical problems can occur, such as failure to converge on a solution, or emergence of excessively high coefficient of variations for the estimated values. A traditional algorithm often used in parameter estimation problem is Levenberg-Marquardt, which is the most popular gradient-based method, converging quadratically to the local minimum [START_REF] Marquardt | An algorithm for least-squares estimation of non-linear parameters[END_REF]. This algorithm was used since the beginning in the MINMOD tool for Minimal Model parameter estimation [START_REF] Bergman | Quantitative estimation of insulin sensitivity[END_REF][START_REF] Pacini | MINMOD: a computer program to calculate insulin sensitivity and pancreatic responsively from the frequently sampled intravenous glucose tolerance test[END_REF].

Apart from this traditional approach other strategies were tested ever since. In particular, Bayesian techniques showed to offer particular advantages in the estimation of insulin sensitivity with the Minimal Model [START_REF] Cobelli | Minimal model SG overestimation and SI underestimation: improved accuracy by a Bayesian two-compartment model[END_REF][START_REF] Agbaje | Bayesian hierarchical approach to estimate insulin sensitivity by minimal model[END_REF][START_REF] Vicini | The iterative two-stage population approach to IVGTT minimal modeling: improved precision with reduced sampling. Intravenous glucose tolerance test[END_REF][START_REF] Andersen | A population-based Bayesian approach to the minimal model of glucose and insulin homeostasis[END_REF], thus providing improvements in the model estimation success rate and in the parameters precision. Recently, Pillonetto et al. [START_REF] Pillonetto | Minimal model S I =0 problem in NIDDM subjects: nonzero bayesian estimates with credible confidence intervals[END_REF] developed a Bayesian parameter estimation strategy for the Minimal Model based on Markov chain Monte Carlo approach. Bayesian estimation technique is based on the incorporation of a priori knowledge into the estimation process and, as noted by Godsland [START_REF] Godsland | The minimal model: an evolving methodology[END_REF], it could be particularly efficient with the Minimal Model, since the knowledge about insulin sensitivity is notable. However, the Bayesian approach may have some drawbacks, such as the requirement for remodelling an entire population dataset in the presence of any a posteriori changes to an individual of the population [START_REF] Agbaje | Bayesian hierarchical approach to estimate insulin sensitivity by minimal model[END_REF][START_REF] Godsland | The minimal model: an evolving methodology[END_REF].

A possible alternative is thus represented by the Genetic Algorithms, GAs. In this study, we implemented a new approach for the estimation of Minimal Model parameters based on GAs, with 150 chromosomes in the initial population and 10000 generations. The modest number of parameters to be estimated, together with the specific genetic operators applied (i.e., "cross-over"

and "mutation" operators), made consistent the choice adopted for the value of the population size [START_REF] De | Analysis of the behaviour of a class of genetic adaptive systems[END_REF].

Beyond the described numerical difficulties in the parameter estimation, another problem exists that limits the usability of Minimal Model in a clinical setting: in several cases the intervention of an operator specifically trained in the model use is necessary to obtain reliable results [START_REF] Hayes | A strategy to enhance the usability of the minimal model as used with the intravenous glucose tolerance test in diabetes research and clinical management[END_REF]. In particular, the choice of the weighing scheme for the first glucose samples of the IVGTT may result to be critical. Based on our experience in Minimal Model analysis, we identified an automated weighting scheme procedure that performed well in the majority of cases, thus avoiding manual intervention of the operator. Moreover, improper choices of the initial parameter values may lead in some cases to unreliable solutions. GAs automatically solved this problem, because they do not require fixing initial values for the parameters but only their limits of existence. Our approach based on automated weighting scheme and GAs for numerical parameter estimation was termed GAMMOD. To the best of our knowledge, no other study applied GAs to the Minimal Model analysis.

Figure captions

Figure 1 -

 1 Figure 1 -Mean model fit (solid curve) and corresponding 95% confidence interval curves (dotted

Figure 2 -

 2 Figure 2 -Comparison of the two methods for the estimation of insulin sensitivity, S I [10 -4 µU ml -1 min -1 ]. The graph displays the scatter diagram, the Deming regression line (solid line) and the

Figure 3 -

 3 Figure 3 -Comparison of the two methods for the estimation of glucose effectiveness, S G [min -1 ].

Clinical Science Immediate Publication. Published on 11 Sep 2006 as manuscript CS20060203

Table 2

 2

		GAMMOD	MINMOD
						Absolute	Absolute
		S I [10 -4 µU ml -1 min -1 ]	S G [min ] -1	S I [10 -4 µU ml -1 min -1 ]	S G [min ] -1	difference in S I [%]	difference in S G [%]
	MEAN	3.86 [9.7]	0.0236 [8.5]	4.33 [17.2]	0.0229 [9.0]	12.1	10.3
	SE	0.19 [1.1]	0.0005 [0.2]	0.20 [2.4]	0.0005 [0.3]	1.1	1.0

Acknowledgements

The IVGTT data come from cooperation between ISIB-CNR and the Department of Internal Medicine III, Division of Endocrinology and Metabolism, University of Vienna. The study was partially supported by a grant from Regione Veneto (DGR 2702/10-09-04) and by the Austrian "Nationalbank Jubiläumsfonds": grant 11198. Umberto Morbiducci wishes to thank Professor Enrico Primo Tomasini for the support to the research activity 13 Clinical Science Immediate Publication. Published on 11 Sep 2006 as manuscript CS20060203 Copyright 2006 The Biochemical Society

Results obtained by the GAMMOD approach were compared to those obtained by the traditional MINMOD approach. The two methods provided very similar estimations for both insulin sensitivity and glucose effectiveness. The difference in the estimations by the two methods is within the precision that is expected for such kind of metabolic parameters. Moreover, such differences are probably of no clinical relevance. As regards the insulin sensitivity index (the parameter of major interest), it is worth noting that the differences between the two methods remain almost the same in any interval of physiological relevance. In fact, the 2.8 • 10 -4 µU ml -1 min -1 value can be assumed as a threshold between impaired and normal insulin sensitivity [START_REF] Kautzky-Willer | Increased intramyocellular lipid concentration identifies impaired glucose metabolism in women with previous gestational diabetes[END_REF], and in the interval 0-2.8 for S I from MINMOD the absolute difference is 12.2±1.9 % (55 subjects); in the interval 2.8-6.9, where 6.9 can be assumed as the threshold over which a subject is certainly not insulin resistant [START_REF] Beard | The insulin sensitivity index in nondiabetic man. Correlation between clamp-derived and IVGTT-derived values[END_REF],the absolute difference is 11.9±1.6 % (90 subjects); for values higher than 6.9 the absolute difference is 12.7±2.8 % (25 subjects). Therefore, no clinically relevant differences are observed in the whole S I range. It must also be noted that some indicators suggest that GAMMOD is performing even better that MINMOD, despite the fact that it does not require manual intervention: in fact, both the coefficient of variations of the estimated parameters and the error of fit are generally lower.

Improved goodness of fit was already observed in GAs applied to modeling in glucose metabolism, i.e. a model of insulin secretion and kinetics based on oral glucose tolerance test [START_REF] Morbiducci | Genetic algorithms for parameter estimation in mathematical modeling of glucose metabolism[END_REF]. The only drawback of GAMMOD compared to MINMOD is the higher computational cost; however, since no real-time results are required, this is not a serious limitation.

In conclusion, we developed a new approach for parameter estimation in the Minimal Model based on automated weighting scheme and Genetic Algorithms. Our approach provided reliable estimation of the model parameters and improved the usability of the model, thus facilitating its further diffusion and application in a clinical setting.

Table legends

Table 1 -Values for S G , p 2 , S I , G 0 obtained using GAMMOD implementation

Mean values for model parameters estimated according to the GAMMOD approach, together with the minimum and maximum individual values obtained in the investigated population. The precision of estimates, expressed in terms of coefficient of variation, CV (%), is also shown for each parameter estimate.


MD Ewoud Ter Avest
email: e.teravest@aig.umcn.nl

MD Allan D Sniderman

Sebastian Jh Bredie

MD Albert Wiegman

Anton Fh Stalenhoef

MD Jacqueline De Graaf

MD Sebastian J H Bredie

MD Anton F H Stalenhoef

Effect of aging and obesity on the expression of dyslipidemia in children from families with familial combined hyperlipidaemia

Keywords: Familial combined hyperlipidemia, apolipoprotein B, children, ageing, obesity

Introduction

Familial combined hyperlipidemia (FCH) is the most common of the familial atherogenic dyslipoproteinemias with a prevalence of up to 5.7 % in the general population [START_REF] Hopkins | Coronary artery disease risk in familial combined hyperlipidemia and familial hypertriglyceridemia: a case-control comparison from the National Heart, Lung, and Blood Institute Family Heart Study[END_REF]. The lipid profile of FCH is complex and affected subjects may present with hypercholesterolemia, hypertriglyceridemia or mixed hyperlipidemia; worse yet, the lipid profile may vary over time in the same subject. Thus, based on lipids, the hallmarks of FCH are variability amongst subjects and within subjects. Amongst all this variability, increased concentrations of apolipoprotein B (apoB) and small dense low-density lipoprotein (sdLDL) stand out as the two most consistent abnormalities amongst FCH subjects [START_REF] Bredie | Nonobese patients with familial combined hyperlipidemia are insulin resistant compared with their non affected relatives[END_REF][START_REF] Bredie | Metabolic and genetic aspects of familial combined hyperlipidaemia with emphasis on low-density lipoprotein heterogeneity[END_REF][START_REF] De Graaf | Defects of lipoprotein metabolism in familial combined hyperlipidaemia[END_REF][START_REF] De Graaf | Metabolic pathogenesis of familial combined hyperlipidaemia with emphasis on insulin resistance, adipose tissue metabolism and free fatty acids[END_REF][START_REF] Veerkamp | Nomogram to diagnose familial combined hyperlipidemia on the basis of results of a 5-year follow-up study[END_REF].

These abnormalities might result from a decreased fractional catabolic rate of apoB-containing particles [START_REF] Aguilar-Salinas | A familial combined hyperlipidemic kindred with impaired apolipoprotein B catabolism. Kinetics of apolipoprotein B during placebo and pravastatin therapy[END_REF], but the majority of studies show that the increased concentrations of apoB and the presence of sdLDL are rather a consequence of increased secretion of very low density lipoprotein (VLDL) particles [START_REF] Kissebah | Integrated regulation of very low density lipoprotein triglycerides and apolipoprotein B kinetics in man: normolipidemic subjects, familial hypertriglyceridemia and familial combined hyperlipidemia[END_REF][START_REF] Kissebah | Low density lipoprotein metabolism in familial combined hyperlipidemia[END_REF][START_REF] Venkatesan | Stable isotopes show a direct relation between VLDL apoB overproduction and serum triglycerides levels and indicate a metabolically[END_REF][START_REF] Teng | Metabolic basis of hyperapobetalipoproteinemia: turnover of apolipoprotein B in low density lipoprotein and its precursors and subfractions compared with normal and familial hypercholesterolemia[END_REF][START_REF] Chait | Very low density overproduction in genetic forms of hypertriglyceridemia[END_REF][START_REF] Cortner | Familial combined hyperlipidemia: use of stable isotopes to demonstrate overproduction of very low density lipoprotein apolipoprotein B by the liver[END_REF]. However, the specific abnormalities responsible for an increased secretion of VLDL remain unknown. One hypothesis is that increased hepatic free fatty acid (FFA) flux due to increased transmembrane adipose tissue FFA flux is responsible [START_REF] Bredie | Nonobese patients with familial combined hyperlipidemia are insulin resistant compared with their non affected relatives[END_REF]. As upper body adipose tissue mass increases, so does transmembrane adipocyte FFA flux and therefore this model predicts agerelated expression of the disorder. A second model posits that a primary disorder in regulation of hepatic sterol balance could be responsible for increased VLDL secretion in which case expression should not be age-dependent [START_REF] Cianflone | Regulation of apoB secretion from HepG2 cells: evidence for a critical role for cholesteryl ester synthesis in the response to a fatty acid challenge[END_REF][START_REF] Watts | Direct correlation between cholesterol synthesis and hepatic secretion of apolipoprotein B-100 in normolipidemic subjects[END_REF]. The first model is more consistent with a phenotype of hyperTG-hyperapoB with increased secretion of relatively triglyceride-rich VLDL, the second with a phenotype of normoTG-hyperapoB and increased secretion of relatively cholesterol-rich VLDL. However, neither model alone would explain the heterogeneity of the lipid phenotypes in FCH.

The objective of the current study was to determine whether we could find evidence for one or the other of these models, one reflecting the fundamental genetically determined metabolic abnormality, the other reflecting the impact of age-related factors such as expansion of adipose tissue mass. Therefore, we explored the phenotype expression of FCH in young adults recruited from families affected with FCH. Moreover, the effect of time and subsequent increases in weight on the phenotype expression in these subjects was determined in a 5-year follow-up study.

Methods

Study population

The study population consisted of kindred of FCH families with known FCH recruited in 1994, 1999 or 2004 and followed up in 1999 and/or 2004 [START_REF] Veerkamp | Nomogram to diagnose familial combined hyperlipidemia on the basis of results of a 5-year follow-up study[END_REF][START_REF] Veerkamp | Diagnosis familial combined hyperlipidemia based on lipid phenotype expression in 32 families: results of a 5-year follow-up study[END_REF][START_REF] Bredie | Apolipoprotein E polymorphism influences lipid phenotype expression, but not the low density lipoprotein subfraction distribution in familial combined hyperlipidemia[END_REF]. All FCH kindreds were recruited by family studies of probands attending the out-patient clinic of the University Medical Centre Nijmegen.

Families were included when, besides a proband presenting with combined hyperlipidemia, at least 1 first-degree relative presented with hypertriglyceridemia (HTG), hypercholesterolemia (HC), or a combined hyperlipidemia. Furthermore, at least the proband or one of the first-degree relatives had to have cardiovascular disease (CVD) before the age of 60 years. Families were excluded when a secondary cause of the hyperlipidemia was diagnosed in the proband (ie, diabetes mellitus, hypothyroidism, and hepatic or renal impairment). None of the probands was homozygous for the apo E2 allele, and none of the probands or first-degree relatives had tendon xanthomas. All subjects in this study were Caucasian.

On every visit, all subjects filled in a questionnaire about their previous medical history, medication use, smoking and drinking habits. Cardiovascular disease was defined by stroke, transient ischaemic attack, myocardial infarction, angina pectoris and/or peripheral vascular disease. On all occasions, weight and height were measured and body-mass index (BMI) was calculated as body weight (in kilograms) divided by the square of height (in meters). Furthermore, venous blood was drawn by venipuncture after an overnight fast and after withdrawal of lipid lowering medication (if used) for 4 weeks. A total of 194 subjects younger than 25 years were recruited from 29 different families (6.6 subjects/family) with an average family size of 30 subjects/family. These patients were seen on one or more visits, and for this group, associations between age, apoB, TG, LDL-subfraction profile and BMI were determined. Associations between BMI, apoB and age were also determined in a control group of 177 healthy, unaffected siblings from children from families with a diagnosis of heterozygous familial hypercholesterolemia (FH). In all these subjects FH diagnosis was excluded by molecular diagnosis: none of the subjects tested positive for LDL-receptor gene mutations or apolipoprotein B mutations. FCH-family subjects were subdivided into two different phenotypes according to their apoB level at their recruitment visit. They were classified as "normoapoB" when their apoB was < 75 th percentile, adjusted for age and gender, or they were classified as "hyperapoB" when their apoB level exceeded the 75 th percentile (reference data obtained from the NHANES III) [START_REF] Bachorik | Apolipoprotein B and AI distributions in the United States, 1988-1991: results of the National Health and Nutrition Examination Survey III (NHANES III)[END_REF]. The 75 th percentile of apoB was chosen since this represents a level of 1200 mg/l in adults, which is the level at which subjects were separated and analyzed with regard to their cardiovascular risk in the Framingham Study [START_REF] Lamarche | Small, dense low-density lipoprotein particles as a predictor of the risk of ischemic heart disease in men. Prospective results from the Quebec Cardiovascular Study Circulation 95, 69-75. Reference intervals for plasma apolipoprotein B determined with a standardized commercial immunoturbidimetric assay: results from the Framingham Offspring Study[END_REF] and the Quebec Cardiovascular Study [20]. Furthermore, it is the level which Veerkamp et al.

Results

Characteristics of young adults from FCH-families stratified by apoB concentration

The subjects with a normoapoB phenotype (n=135) were significantly younger than the subjects with a hyperapoB phenotype (n=59) (18.9±4.0 years compared to 20.9±3.4 years, p<0.001). Gender distribution was equal across both groups, with 53% women in the normoapoB group vs 55% in the hyperapoB group. Clinical and biochemical characteristics for both groups are shown in table 1. The subjects in the hyperapoB group had a significantly higher BMI than the normoapoB group. In addition, subjects with a hyperapoB phenotype had a significantly more atherogenic lipid profile. TC, TG, LDL-C, VLDL-C and VLDL-TG concentrations as well as the VLDL-C/TG ratio were all significantly increased, while HDL-c concentrations were decreased. A mean K-value of -0.18 reflects the presence of small dense LDL within the hyperapoB group. Although HOMA-index was higher in the hyperapoB-group, this difference reached no statistical significance.

Effect of age on plasma lipids, (apo)lipoproteins and body mass index

As would be expected, in the total group of subjects recruited from FCH families, there was a significant positive correlation between age and BMI (r=0.51, p<0.0001). When subjects were analyzed after stratification for age (<20 years>), a significant association was found between age and BMI for subjects aged <20 years (r=0.49, p<0.0001), but not for subjects aged >20 years (r=0.09, p=NS). The significant positive association in subjects aged <20 years however, was not specific for subjects recruited from FCH-families. In the control group of unaffected FH-family offspring (aged 12-19 years, 46% women, mean plasma apoB concentration 820±181 mg.l -1 , mean plasma TG concentration 0.84±0.41 mmol.l -1 , mean BMI 20.1±3.4 kg.m -2 ) we found a similar significant association between BMI and age (r= 0.37, p<0.0001, Figure 1). When the FCH subjects were stratified according to their plasma apoB level, there was a significant association between age and BMI in both groups (normoapoB: r = 0.45, p<0.001, hyperapoB: r = 0.47, p<0.001, Figure 1).

In the FCH population, a significant positive correlation was also present between age and apoB (r=0.45, p<0.001) and this association remained significant when subjects were analyzed after stratification according to their plasma apoB level (normoapoB phenotype: r = 0.45, p<0.001, hyperapoB phenotype: r = 0.47, p<0.001, Figure 2). The association between age and apoB was strongest in subjects aged<20 years (r=0.31, p=0.003) whereas there was no significant association between age and apoB in subjects aged >20 years (r=0.18, p=NS). Again, the relation between age and apoB in subjects aged <20 years was not specific for subjects from FCH-families: a significant association was also found in the control group of unaffected FH-family offspring (r=0.21, p=0.01, Figure 2). In both the normoapoB-group and the hyperapoB group, plasma TG concentrations were associated positively with BMI (respectively r=0.29, p<0.001 and r=0.44, p<0.001), and this was also the case for our control group unaffected siblings of children with FH (r=0.32, p<0.001).

When the subgroup with a hyperapoB-phenotype (n=59) was analyzed separately, it appeared that 53% of the subjects had plasma triglycerides <1.5 mmol.l -1 , which is roughly equivalent to TG-values <75 th percentile adjusted for age and gender according to the population based PROCAM-study cohort [START_REF] Assmann | Results and conclusions of the prospective cardiovascular Munster (PROCAM) study. Lipid metabolism disorders and cardiovascular disease[END_REF]. The vast majority (73%) of these subjects had large buoyant LDL, reflected by a K-value >-0.10 (Figure 3). On the other hand, 80% of the subjects with TG-levels >1.5 mmol.l -1 had sdLDL, as reflected by a K-value <-0.10. However, under the age of 20 years, the presence of TG-levels > 1.5 mmol.l -1 and/or the presence of sdLDL was observed only sporadically. The presence of sdLDL in subjects with a hyperapoB phenotype correlated negatively with age (r = -0.46, p<0.0001). Thus, plasma apoB levels were increased before plasma triglycerides increased or sdLDL was present.

The early rise in plasma apoB levels in subjects under 20 years comes with an early rise in VLDL-c concentrations (0.38±0.16 vs 0.23±0.13 mmol/l, p<0.05 hyperapoB vs normoapoB) and an elevated VLDL-c/TG ratio (0.34±0.07 vs 0.28±0.08, p<0.05 hyperapoB vs normoapoB) as well. Older hyperapoB subjects (>20 years) exhibit even higher VLDL-c concentrations than younger hyperapoB subjects (<20 years) (0.74±0.47 vs 0.38±0.16 mmol/l). This is a reflection of the presence of an increased number of more VLDL-particles rather than being a reflection of a change in composition of these particles, since they have higher apoB levels than younger hyperapoB subjects (1175±146 vs 1018±109 mg/l, p<0.01) but not a higher VLDL-c/TG ratio (0.36±0.08 vs 0.34±0.07, p=NS).

Effect of 5-year follow-up on lifestyle variables

At the time of their recruitment, mean age in the normoapoB group was 19.4± 3.7 years, and in the hyperapoBgroup 20.9±3.8 years (p=0.09). None of the subjects used lipid-lowering medication. Seven out of 23 subjects (30%) smoked cigarettes in the hyperapoB group compared to 23 out of 62 (37%) in the normoapoB group (p=0.48). The number of subjects using >1 alcoholic beverage/day (two in the normoapoB-group, and one in the hyperapoB-group) was not significantly different (p= 0.29). None of the participants had any past or present history of cardiovascular disease at the time of their recruitment.

After a 5 year follow-up, only one subject in both groups used lipid-lowering medication. The percentage subjects consuming >1 alcoholic beverage/day was significantly higher than at the recruitment visit (p<0.001), but no significant differences across the groups were observed. The percentage subjects smoking cigarettes did not change significantly compared to recruitment (p=0.87). After 5-year follow-up still none of the subjects had any past or present CVD.

Effect of 5-year follow-up on plasma lipid and (apo)lipoprotein concentrations and body mass index

Table 2 shows the baseline BMI, lipid and (apo)lipoprotein concentrations and HOMA-indices for the 23 subjects with a plasma level of apoB>75 th percentile adjusted for age and gender, and for the 62 subjects with a plasma level of apoB< 75 th percentile. In these groups, the same (significant) differences in baseline BMI, HOMA, K-value and lipid and (apo)lipoprotein concentrations between the two groups were observed as presented for the whole group of subjects (n=194) above. A significant effect of 5-year follow-up on BMI was found in the linear mixed model. However, this effect was not group-dependent (i.e. there was no time-to group interaction in the model). In both groups, BMI increased significantly after 5 year follow-up (p<0.001 and p=0.002 respectively). This increase in BMI could be attributed to a mean weight-gain of 5.1 kg (from 64.8 to 70.9 kg) in the normoapoB group and to a weight gain of 4.5 kg (from 79.5 to 84 kg) in the hyperapoB group. The difference in weight between the two groups was substantial both at the beginning and at the end of the 5 year follow-up period.

In both groups, apoB, TC and LDL-C concentrations increased after 5 years of follow-up, but these increases reached statistical significance only in the normoapoB-group ( p=0.02, p<0.001 and p=0.002 respectively). For both ApoB, LDL-C and TC, however, time-to-group interaction was not significant (i.e. the increase in ApoB, TC and LDL-C concentration in the normoapoB-group was not significantly different from the increase in the hyperapoB group). The higher plasma concentrations of TG, VLDL-C, and VLDL-TG as well as the higher VLDL-C/TG-ratio, and the higher HOMA-index in the hyperapoB-group at the recruitment visit remained higher after 5-year follow-up. Both in the normoapoB and in the hyperapoBgroup, only small changes in mean-values were recorded, that reached significance only in the larger normoapoB-group. As a result, there was no significant effect of 5-year follow-up on these parameters using the linear mixed model, nor was there a significant time-to-group interaction for any of these parameters. Since the K-value was determined in 1994 and 1999 only and not in 2004, the number of hyperapoB-subjects with 5-year follow-up data on the K-value (n=10) was too small to draw solid conclusions regarding the effect of 5-year follow-up on the occurrence of sdLDL in this group. In the normoapoB-subjects (n=26) however, we observed a significant decrease in K-value with increasing age (p<0.001).

Factors affecting intra-individual changes in BMI and apoB after 5-year follow-up

with a change in TG of 0.015 mmol/l. Since mean weight change in our study was only 4.8 kg, we could not have expected more than 0.072 mmol/l increase in plasma levels of TG. Our actual data are concordant with this relationship. The same may well be true for VLDL-C. VLDL-TG and HOMAvalues. Alternatively, in a recent publication, Noakes et al. [START_REF] Noakes | Weight loss, diet composition and cardiovascular risk[END_REF] identified dietary composition and physical exercise as significant mediators of the relation between weight gain and plasma lipid and lipoprotein concentrations. Since these parameters were not assessed in the current study, they could have acted as confounders. Beyond these factors, it is worth emphasizing that no simple relationship between BMI and apoB expression should be anticipated. To the extent adipose tissue mass influences the level of plasma apoB, it does so based on its effect on hepatic FFA flux. Lower body adipose tissue is associated with low adipocyte transmembrane FFA fluxes and therefore low hepatic FFA fluxes. By contrast, upper body adipose tissue is associated with higher adipocyte transmembrane FFA fluxes and higher hepatic FFA fluxes. Upper body adipose tissue mass does not expand until the lower body adipose tissue reservoirs are filled [START_REF] Smith | 27,000 participants from 52 countries: a case-control study[END_REF]. This explains why there is a tighter relation between waist-to-hip ratio and plasma apoB, insulin resistance, and the risk of vascular disease than between BMI and these indices [30].

Our study has limitations. Almost all our subjects were post adolescent and follow-up data are available for only a subset and in these only over a relatively short period of time. Nevertheless, we think these data are of interest. In the first place, very limited data are available on the phenotypic expression of FCH in young subjects from affected families, and the current study represents the largest data set available. Second, they confirm our previous studies in adults that plasma apoB is the most reliable marker of affected individuals. Third, they indicate that FCH is expressed relatively early in life. At an average age of 21.9 years, the absolute apoB concentration of the 59 subjects in the hyperapoB group was 1119 mg.l -1 , a value for apoB that is only slightly less than the level (1200 mg.l - 1 ) used in adults to diagnose FCH [START_REF] Veerkamp | Nomogram to diagnose familial combined hyperlipidemia on the basis of results of a 5-year follow-up study[END_REF]. Although TG, VLDL-C and VLDL-TG concentrations as well as the HOMA-index were also elevated in the hyperapoB-subjects, the mean values for these parameters were much lower than the corresponding values found in adult FCH patients [START_REF] Veerkamp | Diagnosis familial combined hyperlipidemia based on lipid phenotype expression in 32 families: results of a 5-year follow-up study[END_REF]. These findings are in line with an earlier (albeit cross-sectional) study demonstrating a high incidence (86%) of elevated plasma apoB in children and young adults from families with FCH, whereas the incidence of the hyperapoB-hyperTG phenotype was only 35% [START_REF] Cortner | Prevalence and expression of familial combined hyperlipidemia in childhood[END_REF].

Taken together, the available data suggests there may be two processes augmenting the elevated plasma apoB levels in FCH. The first would be a primary defect, responsible for the high apoB levels early in life, possibly related to sterol balance within the liver [START_REF] Sniderman | Governance of the concentration of plasma LDL: a reevaluation of the LDL receptor paradigm[END_REF]. Multiple lines of evidence have established that regulation of hepatic sterol content is a fundamental metabolic role for apoB secretion and that the rate of hepatic apoB secretion is directly related to the mass of cholesterol ester on the Copyright 2006 The Biochemical Society mass. It seems possible therefore that the final phenotypic expression of FCH is the interplay between a primary genetically determined abnormality in hepatic sterol balance and the superimposed secondary effects of increased FFA flux resulting from the expansion of upper body adipose tissue mass.

In conclusion:

In the present study we found indirect evidence of a presumably genetically determined increased apoB production rate occurring early in life of FCH-family offspring.

Furthermore, we showed that ageing-related factors such as post maturation increases in adipose tissue mass contributed to an aggravation and/or modulation of this genetically determined apoB overproduction.

  endoplasmic reticulum. Hepatic FFA flux is only one of several determinants of cholesterol ester Clinical Science Immediate Publication. Published on 23 Oct 2006 as manuscript CS20060234

  K-value <-0.10, reflecting the presence of small dense LDL. = subject with K-value >-0.10. Horizontal line denotes TG-concentration of 1.5 mmol.l -1 .

TABLE 1 .

 1 Anthropometric measurements, lipid and (apo)lipoprotein concentrations, and HOMAindices of 194 young-adults recruited from FCH-families, stratified by apoB concentration.

		normoapoB	hyperapoB
		th <75 percentile	th >75 percentile	p	*
		(n=135)	(n=59)
	Age (y)	18.9± 4.0	21.0±3.4	0.001
	BMI (kg.m -2 )	21.2±3.0	24.2±4.1	0.001
	TC (mmol.l -1 )	4.20±0.64	5.51±0.82	<0.001

TABLE 2 .

 2 The effect of 5-year follow-up on anthropometric measurements, lipid-and (apo)lipoprotein-concentrations and HOMA-index of 85 young-adults recruited from FCH-families, stratified by apoB concentration at their recruitment visit.Values are estimated mean ± SD at recruitment visit and mean ± SEM changes after 5-year follow-up.

	Abbreviations as in table 1. = statistically significant change (p<0.05) from recruitment by paired t-
	test.* = p-value for fixed effect of the independent variable "time" (5year follow-up) on the dependent
	variable in the linear mixed model with "age" as a co-variate.

TABLE 3

 3 Spearman's rank correlation coefficients representing the correlation between intra-individual changes in respectively plasma ApoB-level and BMI on the one hand and changes in the plasma concentration of the various (apo)lipoproteins, triglycerides and HOMA-index after 5 year follow-up on the other hand (n=85).

		ApoB	BMI
	BMI, (kg.m -2 )	0.27 *
	LDL-C, (mmol. l -1 )	0.59 ‡	0.05

Clinical Science Immediate Publication. Published on 23 Oct 2006 as manuscript CS20060234 Copyright 2006 The Biochemical Society FIGURE 1.

  Association of BMI with age in 194 subjects recruited from families with FCH (stratified by apoB phenotype) and 177 control subjects. Association of ApoB with age in 194 young adults recruited from families with FCH (stratified by apoB phenotype) and 177 control subjects.

	.
				1800,00
		40,00		1600,00
				1400,00
	BMI (kg.m-2)	30,00	ApoB (mg/l)	1000,00 1200,00
				800,00
		20,00
				600,00
				400,00
		10,00
					10	12	15	18	20	22	25
				10	12	15		18 Age (years) 20	22	25
							Age (years)
	Legend. /	= subject (s) with apoB >75 th percentile adjusted for age and gender,	/	=
	subject (s) with apoB< 75 th percentile adjusted for age and gender, /	= unaffected FH
	Legend: siblings.	/		= subject (s) with apoB >75 th percentile adjusted for age and gender,	/	=
	subject (s) with apoB< 75 th percentile adjusted for age and gender ,	/	= unaffected FH siblings.

FIGURE 2.

22 Clinical Science Immediate Publication. Published on 23 Oct 2006 as manuscript CS20060234 Copyright 2006 The Biochemical Society FIGURE 3.

  Association of plasma triglyceride-concentration with age in 59 young adults with the hyperapoB-phenotype (apoB>75 th percentile for age and gender) recruited from FCH-families. Bredie SJ, Vogelaar JM, Demacker PN, Stalenhoef AF. Apolipoprotein E polymorphism influences lipid phenotypic expression, but not the low density lipoprotein subfraction distribution in familial combined hyperlipidemia. Atherosclerosis 1996 October 25;126(2):313-24. (2) Veerkamp MJ, de Graaf J, Bredie SJ, Hendriks JC, Demacker PN, Stalenhoef AF. Diagnosis of familial combined hyperlipidemia based on lipid phenotype expression in 32 families: results of a 5-year follow-up study. Arterioscler Thromb Vasc Biol 2002 February 1;22(2):274-82.

	Reference List
	(1)

Clinical Science Immediate Publication. Published on 23 Oct 2006 as manuscript CS20060234 Copyright 2006 The Biochemical Society

Acknowledgements

We especially thank the patients who participated in this study. The help off Mrs H. Lemmers, Mrs H. Toenhake-Dijkstra, Ms M. Hectors and Mrs E. Bolat-Elbeyli for technical assistance is gratefully acknowledged. J de G is a clinical fellow of The Netherlands Organisation for Health Research and Development, project registration number 907-00-082. Part of this work was enabled by a grant from the Netherlands Heart Foundation, grant number 2003B057.

The study was carried out in accordance with the declaration of Helsinki of the World Medical Association, and approved by the Medical Ethics Committee

Discussion

To the best of our knowledge, this is the first study that has examined the effect of age and body weight on phenotype expression of FCH in a large cohort of children and young adults recruited from FCH families. We show that an elevated apoB concentration is the first expression of FCH in children and young adults. Furthermore, our data support the hypothesis that age-related factors, such as expansion of adipose tissue mass, contribute to the phenotype expression in patients with FCH.

We found that in FCH plasma apoB, plasma triglycerides, BMI, and LDL-particle size were all associated with age. These associations were independently of the apoB phenotype (normoapoB or hyperapoB). Even more, the associations between age, BMI and apoB were not specific for subjects recruited from FCH-families: similar association, were also present in a control group of unaffected siblings of children with FH.

The majority of subjects with an elevated apoB but younger than 20 years had no elevated plasma triglyceride levels or sdLDL, although these traits are frequently observed in adult FCH patients. This suggests that increased secretion of apoB may be the primary abnormality in FCH. On the other hand, the positive trends between plasma TG concentration and age, between age and BMI and between BMI and plasma triglyceride concentration, suggest that increasing adipose tissue mass is a major driver of plasma triglycerides and that this is a secondary trend affecting the expression of FCH. This is in concordance with our finding that the 59 subjects with a hyperapoB phenotype in our study not only had a higher plasma apoB concentration, but they also had a higher BMI as well as a more atherogenic lipid profile than their 135 normoapoB relatives. The combination of a higher BMI and higher plasma TG levels points to a role of adipose tissue in the pathogenesis of their elevated apoB levels by virtue of an increased hepatic FFA flux.

The results of the 5-year-follow-up were in line with these findings. BMI and apoB increased in both the normoapoB and the hyperapoB groups (although the latter not significantly). Furthermore, the intra-individual increase in BMI after 5-year follow-up appeared to be significantly associated with intra-individual changes in plasma apoB concentration. Of importance, the influence of changes in BMI on levels of apoB was not confined to a specific phenotype. Taken together, these findings favour the hypothesis that increased hepatic apoB secretion in subjects with FCH might be augmented by post-maturational changes in adipose tissue mass.

The observed lack of change in mean plasma concentrations of VLDL-C, VLDL-TG, HOMA-index, TG and apoB after 5-year follow-up might be related to the relatively small increases in mean value of BMI. Datillo et al. [START_REF] Dattilo | Effects of weight reduction on blood lipids and lipoproteins: a meta-analysis[END_REF] showed in a meta-analysis that every kilogram weight-reduction is associated