Lucas Fernández-Campo

María T Grande

Julia Diego

Isabel Fuentes-Calvo

Juan Florencio Macías-Núñez

Angel Sánchez-Rodríguez

Jesús Grande

Luis García-Ortiz

Jose M López-Novoa

Carlos Martínez-Salgado
email: carlosms@usal.es

Juan F Macías-Núñez

José M López-Novoa

Effect of different antihypertensive treatments on Ras / MAPKinases / Akt activation in hypertension and diabetes

Keywords: Hypertension, diabetes, Ras GTPase, ARB, ACEi, diuretics ACEi: angiotensin converting enzyme inhibitors, ARB: angiotensin II receptor blockers, GTP: Guanosine-5'-triphosphate

INTRODUCTION

Ras family of proteins is formed by a group of small GTPases that function as transducers of extracellular signals regulating cell survival, growth and differentiation, and they are well-known by their pro-oncogenic effect [START_REF] Downward | Targeting Ras signalling pathways in cancer therapy[END_REF][START_REF] Colicelli | Human Ras superfamily proteins and related GTPases[END_REF]. Ras GTPases seem to be involved in the development of hypertensive processes, as it has been described by several authors: Muthalif et al. [START_REF] Muthalif | Angiotensin II-induced hypertension: contribution of Ras GTPase/Mitogen-activated protein kinase and cytochrome P450 metabolites[END_REF] indicated that the activation of Ras contributes to the development of angiotensin II (AII)-dependent hypertension and associated vascular pathology; more recently, Benter et al. [START_REF] Benter | Signal transduction involving Ras-GTPase contributes to development of hypertension and end-organ damage in spontaneously hypertensive rats-treated with L-NAME[END_REF] suggested that inhibition of Ras-GTPasemediated signalling can attenuate end-organ damage during severe hypertension and endothelial dysfunction. In hypertensive patients AII promotes the synthesis of the arachidonic acid metabolite 20-hydroxyeicosatetraenoic acid (20-HETE), which contracts blood vessels and causes hyperplasia of vascular smooth muscle cells (VSMC), contributing to an increase in blood pressure [START_REF] Su | Inhibition of renal arachidonic acid omega-hydroxylase activity with ABT reduces blood pressure in the SHR[END_REF]. Moreover, it has been shown that 20-HETE stimulates Ras and MAPK activity, and thus mediate VSMC proliferation through this pathway [START_REF] Muthalif | 20-HETE mediates calcium/calmodulin-dependent protein kinase II-induced MAP kinase activation in VSMC[END_REF]; these authors demonstrated that arachidonic acid metabolites (mainly 20-HETE)-induced MAPK activation through Ras, contributes to the vascular alterations, hypertrophy and hypertension induced by AII in rats [START_REF] Muthalif | Angiotensin II-induced hypertension: contribution of Ras GTPase/Mitogen-activated protein kinase and cytochrome P450 metabolites[END_REF]. Recently, Okada et al. [START_REF] Okada | Angiotensin II type 1 and type 2 receptors reciprocally modulate proinflammatory/ pro-fibrotic reactions in activated splenic lymphocytes[END_REF] have shown that AT1 and AT2 AII receptors reciprocally modulate proinflammatory and pro-fibrotic reactions in lymphocytes. It has been also demonstrated that the antihypertensive treatment with losartan (AII receptor blocker, ARB) reduces DNA synthesis in T lymphocytes [START_REF] Sonmez | Effects of losartan treatment on Tcell activities and plasma leptin concentrations in primary hypertension[END_REF], although the same year Petrov et al. [START_REF] Petrov | Long-term treatment with enalapril or losartan does not show antiproliferative effects in peripheral blood mononuclear cells[END_REF] showed that losartan has no antiproliferative effects on mononuclear cells obtained from peripheral blood. Other authors demonstrated that the stimulation of T [START_REF] Downward | Stimulation of p21ras upon T-cell activation[END_REF] and B lymphocyte antigenic receptors [START_REF] Gold | Intermediary signaling effectors coupling the B-cell receptor to the nucleus[END_REF] induces Ras activation. Recent experiments from Ehrhardt et al. [START_REF] Ehrhardt | Distinct mechanisms determine the patterns of differential activation of H-Ras, N-Ras, K-Ras 4B, and M-Ras by receptors for growth factors or antigen[END_REF] have shown that the stimulation of lymphocyte B and T antigenic receptors induces H-, N-and K-Ras activation, whilst the stimulation of these cells with growth factors as interleukin-3 or epidermal growth factor activates preferentially K-Ras.

Ras activates several intracellular signalling pathways [START_REF] Downward | Targeting Ras signalling pathways in cancer therapy[END_REF][START_REF] Colicelli | Human Ras superfamily proteins and related GTPases[END_REF][START_REF] Dhanasekavan | Signaling by dual specificity kinases[END_REF][START_REF] Kerkhoff | Cell cycle targets or Ras/Raf signallig[END_REF]: mitogenactivated protein kinases (MAPK)/Erk pathway regulates cell growth, differentiation and apoptosis; Erk activates many genes through the stimulation of different transcription factors. Another signalling pathway stimulated by Ras through phosphatidylinositol-3-kinase (PI3K) is the protein kinase B (PKB)/Akt pathway; Akt also regulates the expression of a large group of genes, induces protein synthesis, and protects cells from apoptosis, promoting cell survival; one of the functions of Akt is the phosphorylation of the endothelial nitric oxide synthase (eNOS) [START_REF] Takahashi | Synergistic activation of endothelial nitric-oxide synthase (eNOS) by HSP90 and Akt: calcium-independent eNOS activation involves formation of an HSP90-Akt-CaM-bound eNOS complex[END_REF][START_REF] Simoncini | Interaction of oestrogen receptor with the regulatory subunit of phosphatidylinositol-3-OH kinase[END_REF]. Previous studies from our group in myoblasts showed that transforming growth factor-β1 (TGF-β1) induces extracellular matrix (ECM) synthesis through activation of another MAPK, p38 [START_REF] Rodriguez-Barbero | Transforming growth factor-beta1 induces collagen synthesis and accumulation via p38 mitogenactivated protein kinase (MAPK) pathway in cultured L(6)E(9) myoblasts[END_REF]; moreover, the induction of tubule-interstitial fibrosis by unilateral urethral obstruction is associated to Ras, Erk and Akt activations [START_REF] Rodríguez-Peña | Activation of Erk1/2 and Akt following unilateral ureteral obstruction[END_REF]. Our laboratory has recently shown that in the absence of H-and N-Ras isoforms, fibroblasts synthesize bigger amounts of ECM proteins and show an increase in Akt phosphorylation, suggesting that H-and N-Ras isoforms downregulate ECM synthesis via PI3K/Akt signalling; the lack of both H-and N-Ras isoforms is accompanied with a smaller proliferative rate in response to TGF-β1, together with a decrease in Erk phosphorylation, suggesting that H-and N-Ras isoforms mediate proliferation through MEK/Erk activation [START_REF] Martínez-Salgado | Involvement of H-and N-Ras isoforms in transforming growth factor-β1-induced proliferation and in collagen and fibronectin synthesis[END_REF].

We have studied the involvement of Ras in hypertension and diabetes, evaluating changes in Ras activation and in phosphorylation of their effectors Erk 1/2 and Akt, before and after antihypertensive treatment with ARB, inhibitors of angiotensin converting enzyme (ACEi), and diuretics. The endothelium is probably one of the most important tissues involved in the development of hypertension, but in view of the fact that it is not possible to perform these studies on renal and vascular tissues of hypertensive and diabetic patients, we have used lymphocytes obtained from peripheral blood of these patients. These cells seem to be a suitable model to analyze this intracellular pathways in the development of hypertension: altered T-cell activities and changes in T-cell subset ratios have also been reported in hypertension [START_REF] Sonmez | Effects of losartan treatment on Tcell activities and plasma leptin concentrations in primary hypertension[END_REF], and interstitial infiltration of T cells plays a role in the pathogenesis of salt-sensitive hypertension [START_REF] Rodríguez-Iturbe | Oxidative stress, renal infiltration of immune cells, and salt-sensitive hypertension: all for one and one for all[END_REF]; a very recent study by Crowley et al. [START_REF] Crowley | Stimulation of lymphocyte responses by angiotensin II promotes kidney injury in hypertension[END_REF] in a mouse model of severe hypertension demonstrates the robust contribution of lymphocyte proliferation to the pathogenesis of angiotensin II-induced end-organ injury. On the other hand, lymphocytes have AT1 and AT2 receptors [START_REF] Okada | Angiotensin II type 1 and type 2 receptors reciprocally modulate proinflammatory/ pro-fibrotic reactions in activated splenic lymphocytes[END_REF], and the stimulation of T and B lymphocyte antigenic receptors induces Ras activation [START_REF] Downward | Stimulation of p21ras upon T-cell activation[END_REF][START_REF] Gold | Intermediary signaling effectors coupling the B-cell receptor to the nucleus[END_REF][START_REF] Ehrhardt | Distinct mechanisms determine the patterns of differential activation of H-Ras, N-Ras, K-Ras 4B, and M-Ras by receptors for growth factors or antigen[END_REF].

MATERIALS AND METHODS

Materials and reagents

Phenylmethylsulfonyl fluoride (PMSF), Tween-20, bovine serum albumin, human serum and sodium dodecyl sulphate (SDS) were purchased from Sigma Química (Madrid, Spain). Rabbit anti-mouse Akt 1/2, rabbit anti-rat Erk1, mouse anti-human phospho-Erk and rabbit anti-human eNOS antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit anti-human phospho-Akt antibody was purchased to Cell Signaling Technology (Danvers, MA, USA). Magnesium lysis buffer (MLB) was obtained from Upstate Biotechnology (Charlottesville, VA, USA). Goat anti-rabbit and goat anti-mouse IgG (H+L) horseradish peroxidase (HRP) conjugated antibodies, nitrocellulose membrane and Dc protein assay were from BioRad (Hercules, CA, USA). ECL chemiluminescence Western blotting system and Hyperfilm x-ray film were obtained from Amersham Biosciences (Piscataway, NJ, USA). IMDM medium was from Bio Whittaker Labs (Rockland, ME, USA). Ras-GTPase Chemi ELISA kit was from Active Motif (Rixensart, Belgium). The sterile plastic material used in cell culture was obtained from Nunc (Roskilde, Denmark). Fluoroskan Ascent luminometer was from Thermo Scientific (Waltham, MA, USA). Scion Image software was from Scion Corporation (Frederick, MD USA). All other reagents were of analytical grade and obtained from Sigma Química, Probus (Madrid, Spain), and Merck (Madrid, Spain).

Groups of patients, treatments and extraction of blood samples

These studies were performed in patients with essential hypertension with or without (other than hypertension and diabetes), diabetes. Subjects enrolled in the study from July 2004 to January 2008 were from the Hypertension Unit of the University Hospital of Salamanca, Nephrology Service of the Virgen de la Concha Hospital of Zamora, Health Center of Matilla de los Caños, Salamanca, and Health Center of La Alamedilla, Salamanca, Spain. Altogether, 41 hypertensive and 40 diabetic and hypertensive patients complied with the inclusion/exclusion criteria and were asked to participate in the study. Inclusion criteria were: newly diagnosed essential arterial hypertension (blood pressure: 140/90 mmHg, or 130/80 mmHg in diabetic patients) with (blood glucose > 125 mg/dl) or without diabetes, mainly diabetes mellitus type 2. Exclusion criteria included: previous antihypertensive therapy, serious pathologies (other than hypertension and diabetes), renal or hepatic failure, cardiac congestive insufficiency, therapies with corticosteroids, non-steroid anti-inflammatory drugs and other potential hypertension-inducing drugs, heavy smoking and alcoholism. Demographic data of the patients in each group are shown in Table 1; all recruited patients were overweight (body mass index between 25 and 29.9 kg/m2) (Table 1). The efficacy of every antihypertensive treatment is shown in Table 2. At the beginning of the study, hypertensive patients with diabetes had higher levels of systolic (SAP) and mean arterial pressure (MAP) in comparison with hypertensive patients without diabetes (SAP: 178.26 ± 4.22 mmHg in diabetic patients vs. 158.39 ± 2.62 mmHg in hypertensive patients without diabetes; MAP: 123.08 ± 2.39 mmHg in diabetic patients vs. 115.67 ± 1.73 mmHg in hypertensive patients without diabetes). Every antihypertensive treatment induced similar reductions in blood pressure, although ACEi induced the biggest decrease in hypertensive patients (36 mmHg reduction in SAP, 24 mmHg reduction in MAP) and also in diabetic patients with hypertension (21 mmHg reduction in SAP, 14 mmHg reduction in MAP). The experimental protocol performed was in accordance with the Declaration of Helsinki (2000) of the World Medical Association, and also in agreement with the guidelines and approved by the Ethics Committee of the University Hospital of Salamanca (Spain). Each patient included in the study gave his/her consent to participate after full explanation of the purpose and nature of all procedures used. Groups of patients were as follows: 1. 13 hypertensive patients treated with angiotensin II receptor blockers (ARB, either 600 mg/day eprosartan (n = 9), 50 mg/day losartan (n = 2) or 80 mg/day valsartan (n = 2))

2. 19 hypertensive patients treated with ACEi (either 20 mg/day enalapril (n = 12) or 2 mg/day trandolapril (n = 7))

3. 9 hypertensive patients treated with diuretics (either 2,5 mg/day indapamide (n = 7) or 12,5 mg/day hydrochlorothiazide (n = 2))

4. 23 diabetic hypertensive patients treated with ARB (either 600 mg/day eprosartan (n = 2), 50-100 mg/day losartan (n = 2), 80-160 mg/day valsartan (n = 18) or 150 mg/day irbesartan (n = 1))

5. 9 diabetic hypertensive patients treated with ACEi (either 20-40 mg/day enalapril (n = 4), 2 mg/day trandolapril (n = 3), 20 mg/day lisinopril (n = 1) or 10 mg/day imidapril (n = 1))

6. 8 diabetic hypertensive patients treated with diuretics (either 12,5 mg/day hydrochlorothiazide (n = 6) or 40 mg/day furosemide (n = 2))

Peripheral blood samples (10 ml) were obtained by venipuncture after 12 h fasting, before the treatments (basal) and after 1 week and 1, 2 and 3 months of each antihypertensive treatment.

Extraction of lymphocytes from blood samples and lymphocyte culture

Lymphocytes can be separated from granulocytes and erythrocytes according to their lower densities: they will float on top of a density gradient of Ficoll-Hypaque, whereas granulocytes and erythrocytes will traverse this fluid and collect at the bottom of the tube [START_REF] Harris | Rapid preparation of lymphocytes for tissuetyping[END_REF]. 10 ml of whole blood were centrifuged at 160 g during 5 min at room temperature, and yellow supernatant (approx. 1 ml) containing platelets was removed, and the same volume of room temperature phosphate buffered saline (PBS) was added and mixed. 5 ml of blood were added carefully on top of 4 ml of Ficoll-Hypaque, and tubes were centrifuged at 400 g during 30 min at room temperature; the interface band (which includes the lymphocytes) was aspirated along with no more than 5 ml of fluid above the pellet, and transferred to new tubes; volume was completed with PBS and centrifuged at 400 g during 30 min at room temperature; lymphocyte pellet was washed twice with PBS and centrifuged again. A fraction of the lymphocytes obtained was cultured in IMDM supplemented with 10% human male AB plasma, penicillin (0.66 mg/mL) and streptomycin sulphate (60 mg/mL) [START_REF] Browning | The T cell activation marker CD150 can be used to identify alloantigen-activated CD4+25+ regulatory T cells[END_REF].

Monocytes/macrophages comprise 5% to 20% of the lymphocytes prepared by density gradient centrifugation. To isolate T and B cells population, we exploited the adherence property of monocytes/macrophages by coating plastic tissue culture vessels with serum (as a source of fibronectin and other components of the extracellular matrix); monocytes/macrophages, but not T and B cells, adhered to these surfaces, and we finally isolated T and B lymphocytes which remained in the supernatant.

Cell lysates

Lymphocyte extracts were obtained by cell lysis in magnesium lysis buffer (MLB: 25 mM HEPES, pH 7.5, 150 mM NaCl, 1% Igepal CA-630, 0.25% sodium deoxycolate, 1 mM EDTA) containing 10% glycerol and proteases and phosphatases inhibitors (1 mM phenylmethylsulfonyl fluoride,1 µg/ml leupeptin, 1 µg/ml aprotinin, 1 mM Na 3 VO 4 and 25mM NaF). Cell extracts were maintained at 4ºC for 30 minutes, then centrifuged at 15000 g for 20 min at 4ºC, supernatants collected and protein content of cell extracts was determined by the Lowry assay (Dc protein assay).

ELISA detection of Ras-GTP

Ras-GTP (the active form of Ras) was detected by the Ras GTPase Chemi ELISA kit, which contains a Raf-Ras binding domain (RBD) protein fused to glutathione-Stransferase that is coated onto 96-well, glutathione-coated plate. The activated Ras contained in cellular extracts specifically binds to Raf-RBD, while inactive Ras does not bind. Bound Ras is detected by incubating with a primary antibody that detects H-and K-Ras in human samples. Addition of a secondary antibody conjugated to horseradish peroxidase and developing solution provides a sensitive chemiluminescent readout that is quantified by luminescence in a luminometer. We have followed the instructions indicated by the manufacturer, and luminescence was quantified in a Fluoroskan Ascent luminometer.

Western blot analysis

Fresh lymphocyte extracts were employed in the detection of Akt 1/2, phospho-Akt, Erk 1/2 and phospho-Erk. Equal amounts of proteins (20-30 µg) were electrophoresed in 10% (Akt and phospho-Akt) and 12% (Erk and phospho-Erk) SDS-polyacrylamide gels and transferred to nitrocellulose membranes (0.45 µm). Membranes were blocked with blocking buffer (1-5% bovine serum albumin in 20mM Tris-HCl, 150mM NaCl and 0.1% Tween 20, pH 7.5-8) and incubated with the following antibodies: anti-Akt 1/2 (dilution: 1/1000), anti-Erk1 (dilution: 1/10000), anti-phospho-Erk (dilution: 1/2000), anti-phospho-Akt (dilution: 1/1000). After incubation with the corresponding horseradish peroxidase-conjugated secondary antibody (dilution: 1/10000-1/20000), membranes were finally incubated with a chemiluminescent reagent (ECL detection

Statistical methods

Data are expressed as mean ± standard error of the mean (X ± SEM). The Kolmogorov-Smirnov test was used to assess normality of the data distribution. Comparison of means was performed by one way ANOVA. Statistical differences between groups were assessed by the Scheffe´s test. A "p" value lower than 0.05 was considered statistically significant.

RESULTS

Effect of antihypertensive treatment on Ras activation

We quantified the amount of activated Ras (Ras bound to GTP) in lymphocyte extracts obtained from patients before and after different antihypertensive therapies. ARB treatment in hypertensive patients did not induce any significant change on Ras activation during the three months of the study; ACEi treatment reduced Ras activation after 1 month, but showed no effect in the second month of treatment; however, diuretics induced a sustained increase in Ras activation after 1 week of treatment (figure 1A). In diabetic patients with hypertension, every antihypertensive treatment increased Ras activation in the first week, but these increases disappeared after 1 month of treatment (figure 1B).

Effect of antihypertensive treatment on Erk activation

Therapy with ARB did not induce significative changes in phospho-Erk expression in lymphocytes from hypertensive patients, although there is a slight increase in phospho-Erk expression in the last 2 months of the study; ACEi treatment induced a significative increase in phospho-Erk expression in the first week which disappeared after one month, but it was again increased in the last 2 months of the study; however, diuretic treatment reduced phospho-Erk expression in the last 2 months of the study (figure 2A). In patients with diabetes and hypertension, antihypertensive treatments did not induce significant changes in phospho-Erk expression in lymphocytes, although diuretics and ACEi reduced phospho-Erk expression in the first and third month of treatment, respectively (figure 2B).

Effect of antihypertensive treatment on Akt activation

Treatment with ARB or diuretics did not induce any changes in phospho-Akt expression in lymphocytes from hypertensive patients, with or without diabetes (figure 3); ACEi treatment only induced a reduction in phospho-Akt expression in diabetic patients after 1 month of treatment which disappeared in the second month (figure 3B).

DISCUSSION

We have evaluated the effect of antihypertensive treatments on Ras, Erk 1/2 and Akt activation in hypertensive patients with or without diabetes mellitus, preferably type 2 diabetes, although most patients with this pathology usually take antihypertensive drugs; thus, we were not able to recruit a higher number of patients, due to the exclusion criteria lying in previous antihypertensive therapy; moreover, the inclusion criteria of newly diagnosed essential arterial hypertension reduced the number of available patients who could be included in our study.

Diabetes is the most frequent cause of chronic renal failure, and one of the main characteristics of this pathology is the appearance of hypertension. Ras proteins have a relevant participation in the development of diabetes, as it has been widely described: transgenic mice expressing an H-ras hybrid gene in pancreatic beta cells developed beta-cell degeneration and diabetes [START_REF] Efrat | Diabetes induced in male transgenic mice by expression of human H-ras oncoprotein in pancreatic beta cells[END_REF]; inhibition of signal transduction involving Ras-GTPase can prevent development of diabetes-induced abnormal vascular reactivity in the renal artery [START_REF] Yousif | Signal transduction through Ras-GTPase and Ca2+/ calmodulin-dependent protein kinase II contributes to development of diabetesinduced renal vascular dysfunction[END_REF]; moreover, Ras induction of superoxide activates Erk-dependent fibrosis-stimulatory factor and extracellular matrix gene transcription of mesangial cells in diabetes-induced renal injuries [START_REF] Lin | Ras modulation of superoxide activates ERK-dependent fibronectin expression in diabetes-induced renal injuries[END_REF]; H-Ras activation is also important in the activation of the specific signalling events leading to the accelerated retinal capillary cell apoptosis in hyperglycaemic conditions, suggesting the possible use of H-Ras inhibitors to inhibit the pathogenesis of diabetic retinopathy [START_REF] Kowluru | Small molecular weight Gprotein, H-Ras, and retinal endothelial cell apoptosis in diabetes[END_REF][START_REF] Kowluru | Potential contributory role of H-Ras, a small G-protein, in the development of retinopathy in diabetic rats[END_REF].

The presence and activity of Ras GTPases have been widely described in lymphocytes. Many members of the Ras family of GTPases, notably Ras and Rap1A, and the Rho family GTPases, Cdc42Hs, Rac1, Rac2 and RhoA, are important components of signal transduction pathways used by antigen receptors, cytokine and chemokine receptors to regulate the immune response in lymphocytes [START_REF] Cantrell | GTPases and T cell activation[END_REF]. Transduction of activation signals in T cells involves the redundant participation of the Ras superfamily of GTPases [START_REF] Si | Inhibition of lymphocyte activation and function by the prenylation inhibitor L-778,123[END_REF]. The first step is the TCR ligation, which leads to the activation of the Ras/MAPK signal transduction pathway [START_REF] Downward | Stimulation of p21ras upon T-cell activation[END_REF]. T cell receptor activation leads to phospholipase Cγ-mediated translocation of the nucleotide exchange factor RasGRP1 to the Golgi apparatus where it activates Ras [START_REF] Bivona | Phospholipase Cgamma activates Ras on the Golgi apparatus by means of Ras-GRP1[END_REF]. Recently, Ruiz et al. [START_REF] Ruiz | RasGRF2, a guanosine nucleotide exchange factor for Ras GTPases, participates in T-cell signaling responses[END_REF] showed that RasGRF2, another guanosine nucleotide exchange factor for Ras GTPases, participates in T-cell signalling responses. Ras also initiates phosphatidyl-inositol-3kinase (PI3K) and Akt-mediated signalling pathways in human peripheral blood T lymphocytes [START_REF] Samstag | Ras initiates phosphatidyl-inositol-3-kinase (PI3K)/PKB mediated signalling pathways in untransformed human peripheral blood T lymphocytes[END_REF]. Lymphocyte activation and proliferation induced by interleukin-2 are potently blocked by the Ras prenylation inhibitor L-778,123 [START_REF] Si | Inhibition of lymphocyte activation and function by the prenylation inhibitor L-778,123[END_REF]. A persistent impairment in the activation of Ras has been observed in peripheral blood mononuclear cells from patients with insulin-dependent diabetes mellitus [START_REF] Rapoport | Defective activation of p21ras in peripheral blood mononuclear cells from patients with insulin dependent diabetes mellitus[END_REF].

There are only a few studies quoting the effect of ACEi treatment on lymphocytes. Eiam-Ong et al. [START_REF] Eiam-Ong | Apoptosis of circulating lymphocyte in rats with unilateral ureteral obstruction: role of angiotensin II[END_REF] described that continuous treatment with ACEi could abrogate AII-stimulated circulating lymphocyte apoptosis induced by unilateral ureteral obstruction. Oyama et al. [START_REF] Oyama | Angiotensin converting enzyme inhibitors attenuated the expression of G-protein coupled receptor kinases in heart failure patients[END_REF] showed that ACEi treatment could reduce the level of Gprotein coupled receptor kinase in lymphocytes from patients with congestive heart failure. Our results in lymphocytes from hypertensive patients show that the antihypertensive treatment with ACEi induced a small decrease in Ras activation only in the first month of treatment, but increased phospho-Erk expression in the first week and also in the last 2 months of the study; in diabetic patients with hypertension, ACEi increased Ras activation only in the first week, but reduced phospho-Erk expression in the third month of treatment. It is important to emphasize the different effects of ACEi treatment on Ras activation and phospho-Erk expression in hypertensive patients with or without diabetes: ACEi induced a decrease in phospho-Erk expression in diabetic patients, but increased its expression in hypertensive patients without diabetes. Other authors have recently shown that the antihypertensive effects of ACEi are correlated with inhibition of Ras-regulated Erk activation [START_REF] Hu | Involvement of Ras-regulated myosin light chain phosphorylation in the captopril effects in spontaneously hypertensive rats[END_REF]. Early ACEi treatment in spontaneously hypertensive rats significantly inhibited Ras expression at all ages and decreased phosphorylated Erk in mesenteric arteries [START_REF] Hu | Involvement of Ras-regulated myosin light chain phosphorylation in the captopril effects in spontaneously hypertensive rats[END_REF]; Allard et al. [START_REF] Allard | ACE inhibitor reduces growth factor receptor expression and signaling but also albuminuria through B2-kinin glomerular receptor activation in diabetic rats[END_REF] showed that ACEi treatment significantly reduced the increase in Erk 1/2 glomerular expression that was associated with the early phase of diabetes.

Peripheral blood lymphocytes express amiloride-sensitive epithelial Na + channels (ENaC), and lymphocytes from hypertensive individuals have hyperactive ENaC [START_REF] Carter | Hyperactive ENaC identifies hypertensive individuals amenable to amiloride therapy[END_REF]; these authors also showed that specific inhibition of hyperactive ENaC with the diuretic amiloride ameliorate the hypertension. Feig et al. [START_REF] Feig | Lymphocyte membrane sodiumproton exchange in spontaneously hypertensive rats[END_REF] suggest that lymphocytes from spontaneously hypertensive rats have a greater capacity for sodium uptake through the sodium-proton exchanger in comparison with normotensive strains, and such an increased capacity has a pathophysiological role in genetic hypertension. Other studies showed that other diuretics (furosemide and chlorothiazide) reduce potassium concentrations in lymphocyte obtained from hypertensive patients [START_REF] Abraham | Influence of chronic diuretic therapy on serum, lymphocyte and erythrocyte potassium, magnesium and calcium concentrations[END_REF]. In our experimental model, antihypertensive treatment with diuretics have a different effect on Ras activation than ACEi treatment: Ras activation was significantly increased in the last 2 months of treatment, but in diabetic patients this increase was only observed in the first week; however, phospho-Erk expression was reduced in the last 2 months, but in hypertensive patients with diabetes its expression was reduced only after the first month of treatment. Our findings are in concordance with the studies of Panet et al. [START_REF] Panet | Na+/K+/Cl-cotransporter activates MAPkinase cascade downstream to protein kinase C, and upstream to MEK[END_REF], showing that the loop diuretics bumetanide and furosemide, specific inhibitors of the Na + /K + /Cl -cotransporter (NKCC1), inhibit Erk phosphorylation in Balb/c 3T3 fibroblasts; furthermore, these authors also described that the phosphorylation of Erk 1/2 in human peripheral blood lymphocytes was inhibited by bumetanide [START_REF] Panet | Na(+)/K(+)/Cl(-) cotransporter activates mitogen-activated protein kinase in fibroblasts and lymphocytes[END_REF]. As we did before with ACEi, we have to stress the different effects induced by diuretics treatment in hypertensive patients with or without diabetes: the increase in Ras activation after 2 months disappeared in hypertensive patients with diabetes, and the reduction in phospho-Erk expression was not present after 2 months in diabetic patients. It is also important to notice that although there is an increased expression of Ras-GTP in hypertensive patients, the levels of phospho-Erk expression are reduced; Masuelli and Cutler [START_REF] Masuelli | Increased expression of the Ras suppressor Rsu-1 enhances Erk-2 activation and inhibits Jun kinase activation[END_REF] described that the increased expression of the Ras suppressor Rsu-1 enhances Erk-2 activation in cultured fibroblasts, suggesting that Ras may have an inhibitory effect on Erk phosphorylation in some circumstances. Erk 1/2 inhibits the activity of the Ras activator Sos, and Ras stimulates the PI3K/Akt pathway, which is known to inhibit Raf, and therefore, Erk [START_REF] Kolch | Meaningful relationships: the regulation of the Ras/Raf/MEK/ERK pathway by protein interactions[END_REF]; however, in our experimental model, there were no changes in phospho-Akt activation after treatment with diuretics. Thus, such a direct relationship between Ras-GTP and phospho-Erk is not so clear.

Previous studies by Horiuchi et al. [START_REF] Horiuchi | Fluvastatin enhances the inhibitory effects of a selective angiotensin II type 1 receptor blocker, valsartan, on vascular neointimal formation[END_REF] were undertaken to investigate the potential effect of the cross-talk of the AT1 receptor and a hydroxymethylglutaryl coenzyme A reductase inhibitor (statin, also a Ras prenylation inhibitor) in VSMC; pretreatment of VSMC with fluvastatin significantly inhibited AII-mediated Erk activation; consistent with their in vitro results, phosphorylation of Erk was attenuated by the coadministration of valsartan and fluvastatin even at low doses in vivo, suggesting that the cholesterol-independent inhibition of AT1 receptor-mediated VSMC proliferation by statins may contribute to the beneficial effects of statins combined with ARB on vascular diseases. Continuous treatment with ARB could abrogate apoptosis induced by unilateral ureteral obstruction in circulating lymphocytes [START_REF] Eiam-Ong | Apoptosis of circulating lymphocyte in rats with unilateral ureteral obstruction: role of angiotensin II[END_REF]. However, our experiments showed that treatment with ARB do not seem to influence Ras activation and their main signalling pathways (MAPK-Erk and PI3K-Akt) in human lymphocytes.

Our findings support the notion that in some patients and circumstances as the presence of diabetes, and depending on the hypertensive therapy, small GTP-binding proteins and MAPK may be promising therapeutic targets toward cardiovascular diseases, as it has been recently reviewed [START_REF] Shirai | Small GTP-binding proteins and mitogen-activated protein kinases as promising therapeutic targets of vascular remodeling[END_REF][START_REF] Martínez-Salgado | Involvement of small Ras GTPases and their effectors in chronic renal disease[END_REF]. The design of new pharmacological strategies against these targets may be useful to diminish the damage induced by hypertension and diabetes.

Clinical

  Science Immediate Publication. Published on 30 Jun 2008 as manuscript CS20080119 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20080119 S t a g e 2 ( a ) P O S T -P R I N T

  Clinical Science Immediate Publication. Published on 30 Jun 2008 as manuscript CS20080119 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20080119 S t a g e 2 ( a ) P O S T -P R I N T

Clinical

  Science Immediate Publication. Published on 30 Jun 2008 as manuscript CS20080119 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20080119 S t a g e 2 ( a ) P O S T -P R I N T reagent) and developed signals were recorded on x-ray film (Hyperfilm) for densitometric analysis (Scion Image for Windows).

Clinical

  Science Immediate Publication. Published on 30 Jun 2008 as manuscript CS20080119 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20080119 S t a g e 2 ( a ) P O S T -P R I N T

Clinical

  Science Immediate Publication. Published on 30 Jun 2008 as manuscript CS20080119 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20080119 S t a g e 2 ( a ) P O S T -P R I N T

Figure 1

 1 Figure 1 Effect of antihypertensive treatments (ARB, ACEi, diuretics; see methods section for details) during 3 months on Ras activation (GTP-bound Ras) in lymphocytes from hypertensive patients (A) and from diabetic patients with hypertension (B), evaluated by ELISA. Histograms represent the mean + standard error of the mean (SEM) of 8-23 patients per group, and data are expressed as % over basal (100%). * Statistically significant differences (p<0.05) vs. its respective basal.

Figure 2

 2 Figure 2 Effect of antihypertensive treatments (ARB, ACEi, diuretics; see methods section for details) during 3 months on phospho-Erk expression (GTPbound Ras) in lymphocytes from hypertensive patients (A) and from diabetic patients with hypertension (B), evaluated by western blot. Upper panels in A and B show representative blots of the patients analyzed, performed in similar conditions.Histograms represent the mean + standard error of the mean (SEM) of the optical density of the bands from 8-23 blots per group; data represent the ratio phospho-Erk / total Erk, and are expressed as % over basal (100%). Total Erk was also used as loading control. * Statistically significant differences (p<0.05) vs. its respective basal.

Figure 3

 3 Figure 3 Effect of antihypertensive treatments (ARB, ACEi, diuretics; see methods section for details) during 3 months on phospho-Akt expression (GTPbound Ras) in lymphocytes from hypertensive patients (A) and from diabetic patients with hypertension (B), evaluated by western blot. Upper panels in A and Bshow representative blots of the patients analyzed, performed in similar conditions. Histograms represent the mean + standard error of the mean (SEM) of the optical density of the bands from 8-23 blots per group; data represent the ratio phospho-Akt / total Akt, and are expressed as % over basal (100%). Total Erk was used as loading control. * Statistically significant differences (p<0.05) vs. its respective basal.

  Figure 2

Table 1 Demographic and physical characteristics of 81 hypertensive (41) and diabetic (40) patients before antihypertensive therapy.

 1 Data are expressed as mean ± standard error of the mean. These patients were randomized to treatment with angiotensin II receptor blockers (ARB), angiotensin converting enzyme inhibitors (ACEi) and diuretics and completed the study. BMI: body mass index; D: diabetic patients

		Male female age (years) weight (kg) height (m) BMI (kg/m 2 )
	ARB	7	6	50.12 ± 5.86 73.96 ± 4.06 1.67 ± 0.03 27.70 ± 1.07
	ACEi	13	6	61.89 ± 2.95 78.03 ± 3.24 1.63 ± 0.02 29.07 ± 0.90
	diuretic	3	6	61.50 ± 6.62 71.04 ± 4.17 1.60 ± 0.03 29.53 ± 1.03
	D-ARB	15	8	68.74 ± 2.77 74.17 ± 2.49 1.59 ± 0.01 27.70 ± 1.61
	D-ACEi	8	1	61.50 ± 3.28 77.10 ± 4.24 1.65 ± 0.02 28.54 ± 1.82
	D-diuretic	6	2	69.17 ± 5.00 71.79 ± 3.75 1.60 ± 0.03 28.19 ± 1.14

Clinical Science Immediate Publication. Published on 30 Jun 2008 as manuscript CS20080119 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20080119 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Table 2 Blood pressure characteristics of 81 hypertensive (41) and diabetic (40) patients before (basal) and after antihypertensive therapy (3 rd month

 2

	systolic arterial pressure
			basal			3 rd month
	ARB	SAP 151 ± 5	DAP 93 ± 4	MAP 112 ± 4	SAP 126 ± 5	DAP 80 ± 3	MAP 95 ± 3
	ACEi	165 ± 3	96 ± 2	119 ± 2	128 ± 2	78 ± 2	95 ± 2
	diuretic	155 ± 7	93 ± 4	113 ± 3	125 ± 3	77 ± 3	93 ± 2
	D-ARB	174 ± 5	93 ± 3	120 ± 3	156 ± 7	85 ± 3	109 ± 4
	D-ACEi	178 ± 7	99 ± 5	125 ± 5	157 ± 11	89 ± 4	111 ± 6
	D-diuretic 191 ± 11 100 ± 11	130 ± 5	178 ± 13	94 ± 3 122 ± 5

). Data are expressed as mean ± standard error of the mean, and pressure units are mmHg. ACEi: angiotensin converting enzyme inhibitors; ARB: angiotensin II receptor blockers; D: diabetic patients; DAP: diastolic arterial pressure; MAP: mean arterial pressure; SAP: Clinical Science Immediate Publication. Published on 30 Jun 2008 as manuscript CS20080119 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20080119 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

© 2008 The Authors Journal compilation © 2008 Biochemical Society

ACKNOWLEDGEMENTS

This work was supported by grants from European Commission Research Directorates General (ERG-Mobility-11, #508782), Instituto de Salud Carlos III (Ministerio de Sanidad y Consumo, CP03/00094 and PI041817), Junta de Castilla y León (SA001/C05; SA029A05) and Fundación de Investigación Médica Mutua Madrileña. We thank María Luisa Vivas-Madeira and María Cruz Alonso-Díez from the Hypertension Unit of the University Hospital of Salamanca for their kind colaboration on patients appointment and blood samples extraction.


Juan Marcelo Fernández

Marzo Edir Da Silva-Grigoletto

Juan A Ruano-Ruiz

Javier Caballero-Villarraso

Rafael Moreno-Luna

Isaac Tunez-Fiñana

Inmaculada Tasset-Cuevas

Pablo Pérez-Martinez

José López-Miranda

Francisco Pérez-Jiménez

Javier Caballero- Villarraso

Fructose modifies the hormonal response and modulates lipid metabolism in aerobic exercise after glucose supplementation

Keywords: glucose plus fructose, fructose and hypertriglyceridemia, carbohydrate supplementation, exercise and lipidic metabolism Glu, glucose, GluF, glucose plus fructose, DNL, de novo lipogenesis, VLDL, very low density lipoprotein, HR, Heart-rate, VE, ventilation, VO 2, oxygen consumption, VCO 2, carbon dioxide consumption, PETO

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

The pre-exercise ingestion of carbohydrates is a common dietary supplement that delays fatigue and this is frequently consumed between sportsmen or recreational practitioners [START_REF]American College of Sports Medicine, American Dietetic Association & Dietitians of Canada[END_REF]. However, differences in the mechanisms of absorption and the metabolic pathways taken by the ingested carbohydrate provoke a range of metabolic and hormonal responses that are still partially understood.

Fructose is a monosaccharide that has often been studied as an ergogenic supplement in exercise conditions. In comparison with glucose, pre-exercise ingestion of fructose did not lead to reactive hypoglycaemia and improved performance [START_REF] Okano | Effect of pre-exercise fructose ingestion on endurance performance in fed men[END_REF]. This improvement also has been reported when the fructose one was combined with glucose and ingested during an aerobic exercise [START_REF] Riddell | Substrate utilization during exercise with glucose and glucose plus fructose ingestion in boys ages 10-14 yr[END_REF][START_REF] Jentjens | Oxidation of combined ingestion of glucose and fructose during exercise[END_REF].

Nevertheless, in the studies mentioned before, the authors extensively studied some mechanisms related to improved performance, e.g. exogenous and endogenous rate of carbohydrates oxidation, without a directly analysis of the metabolic effects of glucose plus fructose on insulinaemic and glycaemic response. Moreover, at least during rest condition, has been reported a reduction in the insulinaemic and glycaemic response following the combination of different doses of fructose with high glycaemic index carbohydrates [START_REF] Moore | Acute fructose administration decreases the glycemic response to an oral glucose tolerance test in normal adults[END_REF][START_REF] Heacock | Fructose prefeeding reduces the glycemic response to a high-glycemic index, starchy food in humans[END_REF][START_REF] Wolf | Supplemental fructose attenuates postprandial glycemia in Zucker fatty fa/fa rats[END_REF]; a finding that has not been studied in exercise conditions. Such information would be of great importance, since one of the key objectives in aerobic exercise is a low insulinaemia and the reduction of the vagal alpha-adrenergic stimulus to favour the selective use of substrates [START_REF] Barbe | In situ assessment of the role of beta-1, beta-2 and beta-3 adrenoceptors in the control of lipolysis and nutritive blood flow in human subcutaneous adipose tissue[END_REF][START_REF] Campbell | Regulation of free fatty acid metabolism by insulin in humans: role of lipolysis and reesterification[END_REF][START_REF] Arner | Adrenergic regulation of lipolysis in situ at rest and during exercise[END_REF] and beta-adrenergic stimulation for lipolisis [START_REF] Barbe | In situ assessment of the role of beta-1, beta-2 and beta-3 adrenoceptors in the control of lipolysis and nutritive blood flow in human subcutaneous adipose tissue[END_REF].

On the other hand, there is growing interest in studying the effects on lipid metabolism and insulin resistance following the ingestion of fructose [START_REF] Elliott | Fructose, weight gain, and the insulin resistance syndrome[END_REF][START_REF] Daly | Sugars, insulin sensitivity, and the postprandial state[END_REF]. Stimulation of de novo lipogenesis (DNL) [START_REF] Schwarz | De novo lipogenesis during fasting and oral fructose in lean and obese hyperinsulinemic subjects[END_REF], a defect in the clearance of very low density lipoprotein (VLDL) particles [START_REF] Frayn | Dietary sugars and lipid metabolism in humans[END_REF], or even the esterification of non-oxidized free fatty acids in the liver [START_REF] Chong | Mechanisms for the acute effect of fructose on postprandial lipemia[END_REF] are among the explanations of the hypertriglyceridemia induced by fructose in healthy subjects at rest conditions. Meanwhile, the reduction in insulin sensitivity appears to be dependent on fructose-induced lipid modification [START_REF] Leˆ | Metabolic effects of fructose[END_REF].

Nevertheless, a high variability have been observed by the studies carried out to date, depending in part on the quantity of fructose administered [START_REF] Truswell | Food carbohydrates and plasma lipids-an update[END_REF] and the type and quantity of carbohydrate that accompanied the fructose [START_REF] Heacock | Fructose prefeeding reduces the glycemic response to a high-glycemic index, starchy food in humans[END_REF]. Furthermore, other factors that have been studied less, such as the training status of the subjects [START_REF] Mettler | The influence of the subjects' training state on the glycemic index[END_REF] and exercise performed during the post-absorption phase, might also influence glycaemic and lipid metabolism after fructose ingestion. For this reason, there is no consensus regarding the ingestion of fructose, either alone or in combination with high glycaemic

EXPERIMENTAL METHODS

Subjects

Twenty healthy adult men (mean age, 26±4.8 years) volunteered to participate in the study. All subjects were normoglycaemic and normolipemic, and none had undergone pharmacological treatment or had taken vitamin or mineral supplements in the two months prior to the study. The subjects were aerobically trained, participating in physical exercise sessions more than three times a week. Table 1 shows their anthropometric and biological characteristics and training background.

The aims of the study and the possible risks involved were explained and an informed consent formed was signed by each subject before the start of the study. The ethics committee of the Reina Sofía Hospital in Cordova approved all the procedures employed in the study.

*** TABLE 1 NEAR HERE***

Study design

As a preliminary, the participants were subjected to an anthropometric study, an evaluation of their nutritional state and a pretesting session in order to determine suitable work-loads during the experimental exercise sessions (tests of VO 2 max. and 10RM). The subjects were thereafter randomly assigned to perform two experimental trials, in consecutive order, which consisted of preexercise ingestion of glucose (Glu) or glucose plus fructose (GluF), and a session of moderate aerobic exercise (Figure 1). A one-week washout period followed each experimental trial and the subjects were also instructed to avoid moderate to severe physical exercise during the 24 hours before each intervention. This was controlled by means of an exercise auto-self recorder and by analysing basal concentrations of Srm-creatine kinase, with levels higher than 200 U/l -1 qualifying as an exclusion criterion. The consumption of caffeine, alcohol and carbohydrates with a high glycaemic index was also avoided during the day before each trial.

*** FIGURE 1 NEAR HERE *** Nutritional status and diet

A qualitative-quantitative assessment of the frequency of food intake retrospective to the four weeks before the preliminary evaluations was obtained from each subject by a nutritionist. The composition of the normal diet and consumption of alimentary antioxidants were calculated with the aid of tables of the chemical composition of food. This nutritional information was used to prescribe an isocaloric diet, with a moderate glycaemic load and glycaemic index [START_REF] Liu | Dietary glycemic load assessed by foodfrequency questionnaire in relation to plasma high-density-lipoprotein cholesterol and fasting plasma triacylglycerols in postmenopausal women[END_REF][START_REF] Jenkins | Glycemic index of foods: a physiological basis for carbohydrate exchange[END_REF]. The daily ration of vegetables and fruit was set in accordance with the recommended dietary intakes of ascorbic acid. This diet was followed by the subjects for two weeks before the start of the experimental trials and during the washout periods between the interventions. A 24-hour food consumption diary was completed by the subjects during the week before each experimental trial on days 3 (half week) and 6 (the day before the intervention). The composition of the diet followed by the subjects was calculated and is presented in Table 2.

*** TABLE 2 NEAR HERE*** Pretesting and determination of workload

All the participants performed a progressive endurance test on an ergometer cycle (Ergometrics 800, Ergoline, Barcelona, Spain), in order to calculate the load of the endurance exercise, at the same time of day (10.00 -13.00) and under identical ambient conditions (21 -24 o C, 45 -55% relative humidity). The protocol comprised pedalling for 2 min at 25 W, and for other 2 min at 50 W as a warm-up; afterwards, the load was increased at a rate of 25 W min -1 . Subjects were required to maintain a constant pedalling rate of between 60 and 70 revolutions min -1 . The test came to an end when: 1) the subject decided on his own to end it; 2) he could no longer maintain the minimum pedalling rate of 60 RPM; 3) or the criteria for completion of the exercise suggested by the American College of Sports Medicine had been fulfilled [START_REF]ACSM's Guidelines for exercise testing and prescription[END_REF]. Heart-rate (HR) (beats min -1 ) was recorded continuously throughout the tests and for three minutes post-exercise by means of a 12channel ECG (Viasys™, Pulse Biomedical Inc., PA, USA). Gas exchange data were continuously obtained by means of an automatic breath by breath system (Oxycon Delta, Jaeger™, Höchberg, Germany), which was calibrated before each test to the appropriate ambiental conditions. The following parameters were acquired (average of each 15-sec interval): oxygen and carbon dioxide consumption (VO 2 and VCO 2, in l min -1 STP); ventilation (VE, in l min -1 BPTS), VE/VO 2 , VE/VCO 2 , and end-tidal partial pressure of oxygen and carbon dioxide (PETO 2 and PECO 2 ).

Ventilatory thresholds 1 and 2 (VT1 and VT2) and the work-loads (W) corresponding to them were calculated using the methodology suggested by Davis [START_REF] Davis | Anaerobic threshold: a review of the concept and directions for future research[END_REF]. In brief, VT1 was determined by means of the criteria of an increase in both VE/VO 2 and PETO 2 without a concomitant increase in VE/VCO 2. VT2 was determined by means of the criteria of an increase in both VE/VO 2 and PETO 2 and a drop in PETCO 2. VT1 and VT2 were independently calculated by two observers. In the event of failure to agree, a third observer was consulted.

Supplement and exercise protocol

The subjects met up at the laboratory between 08.00 and 09.00, after 10 to 12 hours of nocturnal fasting. Fifteen minutes before the start of the exercise period, they consumed, in accordance with a cross-randomised protocol, a water-based solution (400 ml) of 50 g of glucose (glucose anhydride C 6 H 12 O 6 99.5%) or 50 g glucose plus 15 g monosaccharide fructose. The 50g of glucose, which requires no hydrolysis for absorption, has been established as the minimal amount of glycaemic carbohydrate in the two trials, with the purpose that this may provide the same threshold for detecting changes in the glycaemic response when fructose was given simultaneously in the GluF trial. For such reason, we intentionally used two non isocaloric supplementations like others studies have done previously [START_REF] Moore | Acute fructose administration decreases the glycemic response to an oral glucose tolerance test in normal adults[END_REF][START_REF] Heacock | Fructose prefeeding reduces the glycemic response to a high-glycemic index, starchy food in humans[END_REF]. The Glu and GluF solutions had a total concentration of 12.5% and 16.5% respectively. The fructose provided 23.07% of the carbohydrates in the combined supplement, and this amount was determined on the basis of earlier reports of its digestibility and absorption in combination with glucose under conditions of rest and exercise [START_REF] Fujisawa | The effect of exercise on fructose absorption[END_REF][START_REF] Riby | Fructose absorption[END_REF]. The specific concentration of fructose (3.75%) was lower than the 10% maximum reported as being capable of being absorbed without risk of gastrointestinal symptoms [START_REF] Skoog | Dietary fructosa and gastrointentinal symptoms: A Review[END_REF]. Under both trial conditions (Glu or GluF), the aerobic exercise was performed on the ergometer cycle utilised in the preliminary tests.

Each subject exercised at an intensity that corresponded to the value of W attained at a point equidistant between VT1 and VT2 during the preliminary aerobic tests. For example, an individual who had reached VT1 and VT2 with loads of 100 W and 200 W respectively would perform the exercise session against a load of 150 W. In addition, for warm-up purposes, during the first and second minute of the exercise sessions we employed loads of 25 W and 50 W respectively; after three minutes we utilised the load calculated during the preliminary test until a total of 30 min of exercise had been accomplished. The pedalling rate was the same as used in the preliminary tests.

Blood and urine samples, and analytical assays

On the morning of each experimental trial, a cannula (Vennflon, 16 G, London, UK) was inserted into the antecubital vein in order to extract blood samples. The samples were drawn immediately before the ingestion of the carbohydrates (t -15 ) and during the two hours of the post-prandial period at intervals of 15, 30 and 60 min, including the exercise period (t o ; t 15 ; t 30 ) and during the recovery phase (t 45 ; t 75 ; t 105 ). A very small volume of sterile saline was infused immediately after the extraction of each sample in order to keep the cannula clear. The blood samples were collected into tubes containing 1 g l -1 EDTA, and were always protected from the light. The tubes were stored on ice and plasma was separated within 30 min of extraction by low-speed centrifugation at 1500 g for 15 min at 4 o C. Glucose concentrations were determined by spectrophotometric methods, using a modular analyzer (ISE-4-DDPPEEPP, Hoffmann-La Roche ® , Basel, Switzerland). Plasma insulin levels were measured by chemioluminiscent microparticle immunoassay (CMIA) using an analyzer (Architect i-4000, Abbott ® , Chicago, IL, USA). Plasma triacylglyceride (TAG) levels were measured by spectrophotometry using a modular analyzer (ISE-4-DDPPEEPP, Hoffman-La Roche ® , Basel, Switzerland). Serum Free Fatty Acids (FFA) concentrations were determined using an enzymatic colorimetric assay (NEFA Kit, Roche ® Mannheim, Germany). The levels of lipoperoxide (LPO) were determined using a commercially available kit (LPO-586, Oxis International ® , Portland, OR, USA). Quantitative determination of oxidised-LDL (ox-LDL) in plasma was performed by enzyme immunoassay using a commercially available kit (Biomedica ® , Vienna Austria). Plasma lactic acid was measured by enzymatic colorimetric assay using an analyzer (Cobas 400, Hoffman-La Roche®, Basel, Switzerland).

The total urine production was also collected into sterile containers before the ingestion of the carbohydrates (t-15 ), at the end of the exercise period (t 30 ) and the end of the recovery period (t 105 ).

The subjects were recommended to drink water ad libitum during the exercise and recovery periods in order to encourage urine production. Urinary concentrations of epinephrine (E), norepinephrine (NE) and creatinine were determined by means of high-performance liquid chromatography (HLPC), using a chromatograph (BioRad ® , Hercules, California, USA) with a reverse-phase column (flow rate of 1 ml•min -1 ) and an electrochemical detector (at 500 mV and 10 nA). Urinary concentrations of E and NE were expressed relative to urinary creatinine (nmol/mmol creatinine). In addition, one day prior to collecting the urine we encouraged the subjects they abstain from bananas, coffee, pineapple and walnuts. We also assured they did not take any of the fallowing drugs: pheothiazin, paracetamol, salsinol, isoproterenol or alpha-methyldopa.

Perceived exertion and heart rate

The rating of perceived exertion (RPE) was recorded according to the Borg perceived exertion scale (CR10) at three different times: mid-exercise, after completion of the exercise [START_REF] Borg | The Borg CR10 scale[END_REF], and half-hour after completion of the exercise (Figure 5). This last recording was used to indicate the subjects' overall perception of the session [START_REF] Day | Monitoring exercise intensity during resistance training using the session RPE scale[END_REF]. The scale had been used by the subjects during their last training year; so there was a good reproducibility and a period for such purpose was not deemed necessary. Although, the HR was continuously recorded during the study (Polar ® 810; Kempele,

RESULTS

Plasma glucose, insulin and urinary catecholamine

Plasma insulin concentrations were higher in the GluF than in the Glu trials during the exercise and recovery phases (t 15 and t 75 ; p<0.05), without differences being found between the two interventions in the glucose concentrations during these phases (Figure 2A-B). Lower urinary levels of E and NE (Figure 3A-B) were found at the end of the exercise phase following GluF than after Glu (t 45 ; p<0.05).

*** FIGURE 2 AND 3 NEAR HERE*** Plasma triacylglyceride and free fatty acid concentrations

Following the ingestion of GluF, TAG levels were significantly higher during the exercise phase (t 15 and t 30 ; p<0.05) and acute recovery phase (t 45 and t 75 ; p<0.05) than following Glu intake (Figure 4A). FFA levels after GluF (Figure 4B) were lower half-way through the exercise phase (t 15 ; p<0.05) and during the recovery phase (t 75 ; p<0.05), but were higher at the end of the exercise phase (t 30 ; p<0.05) in comparison with Glu intake.

*** FIGURE 4 NEAR HERE*** Lipoperoxide and oxidized LDL concentrations

Following the ingestion of GluF, plasma levels of LPO were significantly higher during the exercise and recovery periods (t 15 , t 30 , t 45 and t 105 ; p<0.05), as were those of ox-LDL at the end of exercise (t 30 ; p<0.05) than following consumption of Glu alone (Table 3).

*** TABLE 3 NEAR HERE***

Blood lactate concentrations, perceived exertion and heart rate

No differences between the supplements were observed in the levels of blood lactate during either the exercise or recovery periods. The RPE and HR was significantly less after GluF than after Glu (t 15 ; t 30 and t 60 ; p<0.05) (Figure 5).

DISCUSSION

To the best of our knowledge, this is the first study of healthy sportsmen to demonstrate that the addition of a dose of fructose to the pre-exercise consumption of a glucose supplement produces a cluster of acute metabolic changes during a moderate aerobic exercise and the subsequent recovery period. These fructose-induced metabolic dysregulations consist of a higher level of insulinaemia than following the ingestion of glucose alone, a greater rise in TAG and markers of lipid oxidation, and a lower urinary concentration of catecholamine and plasma FFA during and after the aerobic exercise.

Higher levels of insulin are required for a given glucose concentration when the metabolic clearance of glucose has been reduced [START_REF] Radziuk | Insulin Sensitivity and Its Measurement: Structural commonalities among the methods[END_REF]. In our study, insulinaemia was 37.2% and 25.8% higher after GluF than after Glu alone during both the exercise and recovery periods (t 15 and t 75 respectively), without this being accompanied by differences in glycaemic behaviour between the interventions. These findings may reflect both: an improved tolerance to a higher carbohydrate intake, when an extra dose of carbohydrate was administer like fructose; but also, a lower systemic insulin sensitivity following to that fructose ingestion. Dirlewanger et al. [START_REF] Dirlewanger | Effects of fructose on hepatic glucose metabolism in humans[END_REF] have demonstrated that the acute infusion of fructose in healthy volunteers induces hepatic and extrahepatic insulin resistance by doubling the need for insulin required to maintain a glucose steady state. Has been suggested that an overload of lipid metabolites derived from fructose and the subsequent over-production of reactive oxygen species, able to spread outside the liver, could interfere with the insulin signalling [START_REF] Wei | Hepatospecific effects of fructose on c-jun NH2terminal kinase: implications for hepatic insulin resistance[END_REF]. Mainly in animal models of insulin resistance fructose-induced, has been observed that an impaired oxidative state was accompanied by an inflammatory gene response mediated by activated nuclear factor kappa -B (NFκB); and the participation of inflammatory cytokines such as muscle tumor necrosis factor-alpha (TNF-α) [START_REF] Son | Chronic N-acetylcysteine prevents fructose induced insulin resistance and hypertension in rats[END_REF][START_REF] Armutcu | Thymosin alpha 1 attenuates lipid peroxidation and improves fructose-induced steatohepatitis in rats[END_REF][START_REF] Togashi | The contribution of skeletal muscle tumor necrosis factor-alpha to insulin resistance and hypertension in fructose fed rats[END_REF]. In the present study, both postprandial phenomena, a higher rise in TAG and the oxidative damage on lipids, as shown by LPO and ox-LDL in GluF trial, would suggest a higher insulinaemia mediated by inflammatory mechanisms.

Another metabolic change that has been described after fructose consumption is a rise in TAG levels [START_REF] Leˆ | A 4-wk high-fructose diet alters lipid metabolism without affecting insulin sensitivity or ectopic lipids in healthy humans[END_REF][START_REF] Park | Mechanisms of fructose-induced hypertriglyceridaemia in the rat: activation of hepatic pyruvate dehydrogenase through inhibition of pyruvate dehydrogenase kinase[END_REF]. Elevated levels of FFA and TAG have been reported in human, even 45 min after the ingestion of 50 g of fructose [START_REF] Nuttall | Peripheral Glucose Appearance Rate Following Fructose Ingestion in Normal Subjects[END_REF]. In our study, 30 and 45 min after the ingestion of GluF (half-way through and at the end of the exercise), we observed TAG concentrations that were 38.7 and 34.2% higher than after Glu in the same times. Moreover, the TAG rapidly returned to basal level 45 minutes after its peak level (t75) following to GluF, discarding the hypothesis of a delay in the TAG clearance as one of the possible causes of the hypertriacylglycerolemia since it has been proposed [START_REF] Chong | Mechanisms for the acute effect of fructose on postprandial lipemia[END_REF]. Furthermore, the higher level of insulin found with GluF leads us to believe that the mechanism that could have made the greatest contribution to the rise in TAG, was not a reduced activation of lipoprotein lipase in adipose tissue and the subsequent delay in lipid clearance [START_REF] Brunzell | Evidence for a common, saturable, triglyceride removal mechanism for chylomicrons and very low density lipoproteins in man[END_REF], but rather DNL, a mechanism widely explained after fructose intake [START_REF] Schwarz | De novo lipogenesis during fasting and oral fructose in lean and obese hyperinsulinemic subjects[END_REF][START_REF] Faeh | Effect of fructose overfeeding and fish oil administration on hepatic de novo lipogenesis and insulin sensitivity in healthy males[END_REF]. In these lines, several studies has been reported that the fast arrival of fructose to the liver it could cause an overload of the pentose phosphate pathway leads to the acute expression of lipogenic genes [START_REF] Wei | An acute increase in fructose concentration increases hepatic glucose-6-phosphatase mRNA via mechanisms that are independent of glycogen synthase kinase-3 in rats[END_REF][START_REF] Kabashima | Xylulose-5-phosphate mediates glucose-induced lipogenesis by xylulose-5-phosphate-activated protein phosphatase in rat liver[END_REF]; to the rapid activation of hepatic lipogenesis and the secretion of VLDL via sterol regulatory element-binding protein-1c (SREBP-1c) [START_REF] Miyazaki | Stearoyl-CoA desaturase 1 gene expression is necessary for fructose-mediated induction of lipogenic gene expression by sterol regulatory element-binding protein-1c-dependent and -independent mechanisms[END_REF]. In those conditions, the mechanisms of reduction of the glycaemic response (e.g. the hepatic uptake of glucose and glycogen synthesis) possibly are inhibited, while the gluconeogenic and lipogenic pathways are activated [START_REF] Wei | An acute increase in fructose concentration increases hepatic glucose-6-phosphatase mRNA via mechanisms that are independent of glycogen synthase kinase-3 in rats[END_REF]. The present study was not designed to investigate the molecular mechanisms responsible for the increase of TAG after GluF ingestion. However, the glucose intake, with an extra dose of fructose (a carbohydrate shown like lipogenesis inducer) and the performance of an aerobic exercise, that may have accelerated the arrival of fructose to the portal system, likely they are the key to explain the shown lipidic behaviour.

On the other hand, it is known that the utilization of substrates during aerobic exercise is modulated by autonomic and endocrine control in response to the type and amount of carbohydrate ingested [START_REF] Borer | Hormonal regulation of fuel use in exercise[END_REF]. Although this study does not provide data that describe substrate oxidation, it has been sufficiently reported that fructose by itself [START_REF] Tappy | Comparison of thermogenic effect of fructose and glucose in normal humans[END_REF], as well as, an elevated insulinaemia during exercise [START_REF] Mandarino | Effects of insulin infusion on human skeletal muscle pyruvate dehydrogenase, phosphofructokinase, and glycogen synthase[END_REF] increase carbohydrate oxidation, to the detriment of the lipid contribution to energy consumption. Additionally, in agreement with McLaren et al. [46], we have found that the elevated insulinaemia was accompanied by both; a less concentration of catecholamine in the urinary output from t -15 to t 30 of exercise period and a low availability of FFA in the GluF trial. If a lower activation of the lipolisis followed GluF, as it is suggested by these findings, the addition of a dose of fructose, would avoid an important part of the benefits for the health derived of 30min of aerobic exercise. Conversely, if GluF increased the carbohydrate oxidation rate, as reported by other studies with glucose plus fructose [START_REF] Riddell | Substrate utilization during exercise with glucose and glucose plus fructose ingestion in boys ages 10-14 yr[END_REF][START_REF] Jentjens | Oxidation of combined ingestion of glucose and fructose during exercise[END_REF], the use of this faster available fuel, would explain a minor RPE and HR, without a change on blood lactate. Thus, the improvement in these variables during and after the exercise with GluF intake could increase the capacity to perform the exercise more comfortably, but it would not favour the metabolic benefits aimed with an aerobic exercise. In conclusion, the addition of fructose to the pre-exercise ingestion of a glucose supplement triggers a greater insulin secretion and a lower adrenergic stimulation than the ingestion of glucose alone. This combination of carbohydrates also induces a larger rise in plasma triglycerides, a deterioration of the oxidative state of circulating lipids and the suppression of circulating free fatty acids during the performance of moderate intensity aerobic exercise and the post-exercise recovery phase. Future studies in risk populations (e.g. diabetics, obese) are required, in order to analyse the metabolic effect of supplements containing different doses of fructose in mixed pre-exercise meals or beverages.

Acknowledgments: Supported by grant "Consejería de Turismo, Comercio y Deporte de la Junta de Andalucía" and "Fundación Hospital Reina Sofía Caja Sur". None of the authors had a personal or financial conflict of interest. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Clinical

  Science Immediate Publication. Published on 05 Jun 2008 as manuscript CS20080120 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20080120 S t a g e 2 ( a ) P O S T -P R I N T

Clinical

  Science Immediate Publication. Published on 05 Jun 2008 as manuscript CS20080120 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20080120 S t a g e 2 ( a ) P O S T -P R I N T

Clinical

  Science Immediate Publication. Published on 05 Jun 2008 as manuscript CS20080120 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20080120 S t a g e 2 ( a ) P O S T -P R I N T

  Clinical Science Immediate Publication. Published on 05 Jun 2008 as manuscript CS20080120 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20080120 S t a g e 2 ( a ) P O S T -P R I N T

  Finnland) Clinical Science Immediate Publication. Published on 05 Jun 2008 as manuscript CS20080120 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20080120 S t a g e 2 ( a ) P O S T -P R I N T Statistical analysis Traditional statistical methods were used to calculate means and standard deviations (SD). The normality of the samples was tested using the Shapiro-Wilk test. The effect of the different interventions (Glu and GluF, independent variables) on glycaemia, insulinaemia, E, NE, TAG, FFA, ox-LDL and LPO (dependent variables) was analyzed by analysis of variance (ANOVA) with repeated [2 (group) x 7 (time)] measures. A Sidak correction was used to adjust the p-value in relation to the number of contrasts that were performed. A p<0.05 criterion was adopted for statistical significance; for all the statistical tests we used the SPSS 11.5 package for Microsoft Windows. Clinical Science Immediate Publication. Published on 05 Jun 2008 as manuscript CS20080120 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20080120 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

*** FIGURE 5

 5 NEAR HERE*** Clinical Science Immediate Publication. Published on 05 Jun 2008 as manuscript CS20080120 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20080120 S t a g e 2 ( a ) P O S T -P R I N T

Clinical

  Science Immediate Publication. Published on 05 Jun 2008 as manuscript CS20080120 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20080120 S t a g e 2 ( a ) P O S T -P R I N T by other authors

Clinical

  Science Immediate Publication. Published on 05 Jun 2008 as manuscript CS20080120 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20080120 S t a g e 2 ( a ) P O S T -P R I N T

Clinical

  Science Immediate Publication. Published on 05 Jun 2008 as manuscript CS20080120 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20080120 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

  Figure legends.

Figure 1

 1 Figure 1 Schematic diagram of experimental protocol used in glucose (Glu) or glucose

Figure 2

 2 Figure 2 Plasma glucose (A) and insulin (B) concentrations at rest, throughout exercise and recovery period with the pre-exercise ingestion Glu or GluF. Values are means ± SE; n=20 subjects. There were significant carbohydrate x time interactions for plasma insulin (p=0.003) by repeatedmeasures ANOVA. *Significantly different in Glu compared with GluF, p<0.05.

Figure 3

 3 Figure 3 Urinary epinephrine (A) and norepinephrine (B) concentrations at baseline, end of exercise and recovery period with the pre-exercise ingestion Glu or GluF. Values are means ± SE; n=20 subjects. There were significant carbohydrate x time interactions for urinary epinephrine and norepinephrine (p=0.001 and p=0.02 respectively) by repeated-measures ANOVA. *Significantly different in Glu compared with GluF, p<0.05.

Figure 4

 4 Figure 4 Plasma triglyceride (A) and free fatty acid (B) concentrations at rest and during exercise and recovery period following the pre-exercise ingestion of Glu or GluF. Values are means ± SE; n=20 subjects. There were significant carbohydrate x time interactions for plasma triglyceride and FFA (p=0.001 and p=0.03 respectively) by repeated-measures ANOVA.*Significantly different in Glu compared with GluF, p<0.05.

Figure 5

 5 Figure 5 Rate of perceived exertion, heart rate and blood lactate at rest, middle and end of exercise and its recovery period, for Glu or GluF interventions. Values are means ± SE; n=20 subjects. There were significant carbohydrate x time interactions for RPE (p=0.032) by repeated-measures ANOVA.*Significantly different in Glu compared with GluF, p<0.05.

Clinical

  Science Immediate Publication. Published on 05 Jun 2008 as manuscript CS20080120 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20080120 S t a g e 2 ( a ) P O S T -P R I N T

Clinical

  Science Immediate Publication. Published on 05 Jun 2008 as manuscript CS20080120 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20080120 S t a g e 2 ( a ) P O S T -P R I N T

Table 1

 1 Characteristics and training background of the study subjects. Values are means ± SD.

	Tables
		Variables	Mean	SD
		Age (years)	26	4,85
	Biological	Weight (Kg) BMI (Kg/m 2 )	75 23,45	10,5 1,93
		% Body fat	16,96	3,44
		Glycaemia (mmol/L)	4,19	0,43
		Total Cholesterol (mmol/L)	3,60	0,68
	Biochemical	Triglycerides (mmol/L)	0,70	0,21
		High Density Lipoprotein (mmol/L)	1,37	0,34
		Low Density Lipoprotein (mmol/L)	1,88	0,48
		Years	2,2	1,3
	Total Training	Day/week Sessions/week	5,7 6,3	0,5 1,2
		Min/week	349,7	69,2
	Endurance Training	Sesion/week Min/week	5,4 246,3	0,8 60,6

Table 2

 2 Dietary composition during the week before Glu or GluF trial. Values are means ± SD. Clinical Science Immediate Publication. Published on 05 Jun 2008 as manuscript CS20080120 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20080120

	Dietary Variables	Mean	SD
		Total Calories (kcal/day)	1790,74	263,56
	Caloric Distribution	% Carbohydrate % Protein % Fat	44,56 23,85 31,25	5,89 3,59 5,54
		Energy Density (kcal/g)	1,36	0,21
		Total Carbohydrate (g)	200,01	38,64
	Dietary	Dietary Glycaemic Load	124,41	16,43
	Carbohydrate	Dietary Glycaemic index	63,51	6,85
		Total Fiber (g)	23,24	5,51
	Ascorbic acid	Intake (mg/day) % RDIs	65,54 109,2	26,94 -

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Table 3

 3 Lipoperoxide and Oxidised LDL measured immediately before Glu or GluF supplementation (t -15 ) and at their respective exercise (t 0 , t 15 and t 30 ) and recovery phases (t 45 and t 105 ). Values are means ± SE. * Mean values were significantly different (p <0.05) between the groups. Values adjusted for body mass index (BMI).

	Time, min

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

© 2008 The Authors Journal compilation © 2008 Biochemical Society

S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society