Giulia Dell'omo

Giuseppe Penno

Laura Pucci

Daniela Lucchesi

Carmen Fotino

Stefano Del Prato

Prof Roberto Pedrinelli
email: r.pedrinelli@med.unipi.it

Stefano Del Prato

Roberto Pedrinelli Dipartimento

Cardio Toracico

Dipartimento Di

Endocrinologia E Metabolismo

ACE gene insertion/deletion polymorphism and capillary permeability in hypertension

Keywords: ACE POLYMORPHISM, TRANSCAPILLARY ESCAPE RATE OF ALBUMIN, ALBUMINURIA, ENDOTHELIAL FUNCTION, HYPERTENSION ANGIOTENSIN CONVERTING ENZIME, ACH:ACETYLCHOLINE, AUC

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INTRODUCTION

Consistent evidence supports the role of the Angiotensin I Converting Enzyme (ACE) Insertion/Deletion (I/D) polymorphism in the modulation of urinary albumin excretion (UAE) in non diabetic patients with essential hypertension. Several previous reports [e.g. [START_REF] Pontremoli | The deletion polymorphism of the angiotensin I-converting enzyme gene is associated with target organ damage in essential hypertension[END_REF][START_REF] Kario | Hypertensive nephropathy and the gene for angiotensin-converting enzyme[END_REF][START_REF] Fernandez-Llama | Angiotensin converting enzyme gene I/D polymorphism in essential hypertension and nephroangiosclerosis[END_REF][START_REF] Redon | Influence of the I/D polymorphism of the angiotensin-converting enzyme gene on the outcome of microalbuminuria in essential hypertension[END_REF][START_REF] Pedrinelli | α-adducin and ACE polymorphisms in hypertension. Evidence for a joint influence on albuminuria[END_REF], in fact, associated the presence of the D allele within the ACE gene with higher UAE and more frequent microalbuminuria (MA), a sign of subclinical renal damage and a predictor of atherosclerotic cardiovascular events [START_REF] Pedrinelli | Microalbuminuria, an integrated marker of cardiovascular risk in essential hypertension[END_REF] in hypertension. It is unknown, however, whether that noxious genetic influence on UAE, which may reflect more permeant renal glomeruli, is also accompanied by systemic changes in systemic permeability of capillary endothelium, a key determinant of endogenous defense against vascular injury and atherosclerosis [START_REF] Hinsbergh | Endothelial permeability for macromolecules. Mechanistic aspects of pathophysiological modulation[END_REF][START_REF] Mehta | Signaling mechanisms regulating endothelial permeability[END_REF].

To assess that possibility, we measured the trans-capillary albumin escape rate (TERalb), a measure of the integrity of systemic capillary permeability [START_REF] Parving | Microvascular permeability to plasma proteins in hypertension and diabetes mellitus in man-on the pathogenesis of hypertensive and diabetic microangiopathy[END_REF], and its relationship with the ACE I/D genotype and UAE, in a group of non diabetic, never treated uncomplicated hypertensive men.

To evaluate a possible coexistence of an abnormal function of arteriolar endothelium, we also investigated the association of that genotype with the endothelial-mediated vasodilator response to intra-brachial acetylcholine (ACH), a nitric oxide (NO) releasing agonist [START_REF] Furchgott | The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine[END_REF].

MATERIAL AND METHODS

Subjects

Seventy-nine never treated sedentary, Caucasian, apparently genetically unrelated hypertensive men and 16 normotensive men age-matched as a group participated in the study.

Exclusion criteria were diabetes (fasting plasma glucose levels >125 mg/dl and/or 2-hr postglucose load ≥200 mg/dl), renal insufficiency (serum creatinine >1.4 mg/dl), proteinuria at the dipstick test and impaired cardiac function (ejection fraction <50%). In all, coexisting coronary and vascular disease were excluded through history, physical examination, routine blood chemistry, baseline EKG or, when indicated, treadmill test, echocardiography, carotid and lower limb echo-Doppler sonography.

In accordance with the Declaration of Helsinki and institutional guidelines, the local Ethical Committee approved the protocol and subjects were aware of the investigational nature of the study and agreed to participate after signing a written consent.

MAIN EVALUATION VARIABLES

ACE genotype determination

applied on the arm, 5-6 cm distal to the elbow at a tension sufficient to keep the gauge in the same position throughout the experiment. Patient forearm was kept on a table, slightly flexed and inclined at about 45° to the horizontal plane with the wrist and hand supported by sand bags. One minute before FBF determination, a pneumatic paediatric cuff was placed around the wrist and inflated to suprasystolic arterial blood pressure in order to exclude the hand vascular region. A second cuff was placed proximal to the plethysmograph and automatically inflated to a pressure of 40 mmHg to allow FBF measurement according to the venous occlusion method. BP was measured every 5 minutes throughout the study at the contralateral arm by an automated device (NIBP KO 7267.004, Kontron Instruments). Further details of the method have been published elsewhere [START_REF] Dell'omo | Simvastatin, capillary permeability and endothelial-mediated vasomotion in atherosclerotic, hypercholesterolemic men[END_REF].

Fresh solutions of ACH HCl (Miovisin™, Farmigea, 7.5, 15 and 30 μg/min, five minutes each), a nitric oxide releasing compound [START_REF] Furchgott | The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine[END_REF], and sodium nitroprusside (SNP, Nipride™, Malesci, used as an internal control for NO-independent mechanisms [START_REF] Bohme | Effects of sodium nitroprusside and other smooth muscle relaxants on cyclic GMP-formation in smooth muscle and platelets[END_REF], 0.8, 1.6 and 3.2 μg/min, five minutes each in syringes protected from light through aluminium foil were infused intra-arterially.

Preliminary studies had shown that those infusion periods were sufficient to reach a plateau of FBF response without changes in systemic arterial pressure and contralateral FBF. Local administration of ACH and SNP was preceded and followed by saline infusion (0.6 ml/min). The infusion sequence was randomized and a 30-minute interval was left between the first and second drug administration.

Ancillary parameters

Office systolic and diastolic blood pressure (BP) was the mean of several indirect recordings. 24 hour BP (08 30 -08 00 A.M) was measured through an oscillometric monitor (Diasys Integra, Novacor).

OGTT (75 g glucose) was performed in the morning after an overnight fast. Upon baseline samples, specimens for plasma glucose and insulin were drawn 0. Total, high density (HDL) and low density lipoprotein (LDL) cholesterol, triglycerides were assessed in fasting conditions by enzymatic colorimetric techniques (Roche, Modular system).

Anthropometric measurements (height and weight) were made after each participant had removed his shoes and upper garments. Body weight was measured to the nearest 0.1 Kg on a scale with attached height measure (SECA 207).

Smoking status was defined as active smokers vs non smokers, without distinction between former-and never smokers.

Data processing

Plasma 125 I-albumin concentration (cpm/ml) was plotted on a semi-logarithmic scale, and the transcapillary escape rate (%/h) was calculated from the mono-exponential disappearance rate constant of the 125 I curve from 10 to 60 min. Plasma volume (ml/1.73 m 2 ) was determined by retropolation to zero time of the disappearance curve corrected for the injected dose of tracer obtained by weighing the syringes before and after the injection [START_REF] Nannipieri | Increased transcapillary escape rate of albumin in microalbuminuric type II diabetic patients[END_REF]. UAE (μg/min) was the average of three consecutive overnight collections (mean variation coefficient of the triplets: 22%). MA was defined as UAE ≥15 μg/min, an accepted threshold for overnight collections [START_REF] Jensen | The Copenhagen City Heart Study Group (1993) Urinary albumin excretion in a population based sample of 1011 middle aged nondiabetic subjects[END_REF]. Because of its skewed distribution, albuminuria was log-transformed to normalize its distribution. Creatinine clearance (ml/min x 1.73m 2 ) was calculated according to standard formulae 1 FBF (ml 100 ml -1 forearm volume min -1 ) represents the mean of four-five determinations obtained over the last two minutes of each experimental period. Percent FBF changes from baseline were the evaluation variable.

OGTT data were summarised as the area under curve (AUC, trapezoidal rule) values. Insulin sensitivity was assessed through the homeostasis model assessment of insulin resistance (HOMA-IR) 2 [START_REF] Mather | Repeatability characteristics of simple indices of insulin resistance: implications for research applications[END_REF]. Increasing HOMA-IR values denote progression from normal to impaired insulin [START_REF]Executive Summary of the Third Report of The National Cholesterol Education Program [NCEP] Expert Panel on Detection, Evaluation, And Treatment of High Blood Cholesterol In Adults (Adult Treatment Panel III) (2001)[END_REF] (elevated BP and at least 2 of the following: triglycerides ≥150 mg/dL, HDL cholesterol <40 mg/dL, fasting plasma glucose ≥ 110 mg/dL, BMI >30 Kg/m 2 ).

Statistics

Proper ANOVA models and χ2 statistics tested the statistical significance of continuous and categorical parameters respectively. Intraindividual association between variables was tested by correlation coefficients analysis. The association of TERalb (coded as 1 for upper-quartile values and 0 for the three remaining ones, cut-off: 11.1 %/hr) with the ACE D/I genotype and other continuous and categorical covariates was analyzed by multivariate logistic regression and odds ratios (ORs) and 95% confidence interval (CI)s were used to estimate relative risks. Statistical significance was set at p<0.05. Descriptive statistics were means±SD or geometric means (interquartile range) for skewed parameters.

RESULTS

As a whole, 42 (45%) subjects showed a D/D genotype vs 42 (45%) and 7 (10%) with a I/D and I/I genotype respectively. Allelic distribution was in Hardy-Weinberg equilibrium (chi-square<0.271, df=1, p>0.6) and did not differ by blood pressure status (table 1). Because

TERalb by ACE genotype

Fasting and stimulated glucose and insulin, HOMA index, triglycerides, BMI, UAE was higher and MetS more frequent in hypertensive patients than normotensive controls (table 1). As compared with normotensive controls, TERalb was higher in hypertensive patients, either as a whole (7.4±1.5%/hr, n=16, vs 9.8±2.4%/hr, n=79, p=0.

Pathophysiological implications

Although the mechanistic links between the ACE polymorphism and a remote and complex phenotype such as capillary permeability cannot be elucidated, some plausible pathophysiological inferences may still be drawn from our data. First, whatever the underlying biological mechanism, the unfavorable influence of the ACE D/D genotype should not be seen as the cause but rather as a modulating factor for the defective systemic capillary permeability featuring human hypertension [START_REF] Parving | Microvascular permeability to plasma proteins in hypertension and diabetes mellitus in man-on the pathogenesis of hypertensive and diabetic microangiopathy[END_REF][START_REF] Parving | Transcapillary escape rate of albumin and plasma volume in essential hypertension[END_REF][START_REF] Pedrinelli | Microalbuminuria and transvascular albumin leakage in essential hypertension[END_REF] since irrespective of the ACE genotype, TERalb was higher in hypertensive patients than normotensive controls. One might also wonder whether the influence of the ACE D/D polymorphic extends to normotensive subjects but the limited number of control subjects included in our sample makes this point irrelevant. As a second point, the effect of a series of sensitive parameters for capillary albumin permeation, such as elevated BP [START_REF] Pedrinelli | Microalbuminuria and transvascular albumin leakage in essential hypertension[END_REF] and LDL cholesterol [START_REF] Dell'omo | Simvastatin, capillary permeability and endothelial-mediated vasomotion in atherosclerotic, hypercholesterolemic men[END_REF], hyperinsulinemia [START_REF] Hilsted | Dual effect of insulin on plasma volume and transcapillary albumin transport[END_REF], smoking habits [START_REF] Jensen | Long-term smoking increases transcapillary escape rate of albumin[END_REF], MetS phenotype [START_REF] Dell'omo | Abnormal capillary permeability and endothelial dysfunction in hypertension with comorbid Metabolic Syndrome[END_REF], was likely exclude because of the homogeneous distribution of those parameters across ACE genotypes. Atherosclerotic vascular disease also affects TERalb [START_REF] Pedrinelli | Transvascular and urinary leakage of albumin in atherosclerotic and hypertensive men[END_REF] but our patients were carefully selected for absence of clinical vascular disease. Given the above considerations, other possibilities should be considered and renin angiotensin system activation appears as a plausible candidate mechanism. In fact, D/D homozygosis associates with higher circulating and tissue ACE levels [START_REF] Rigat | An insertion/deletion polymorphism in the angiotensin I-converting enzyme gene accounting for half the variance of serum enzyme levels[END_REF], the rate limiting step in biologically active angiotensin II production [START_REF] Muller | Vascular angiotensin-converting enzyme expression regulates local angiotensin II[END_REF], and also stimulates its local production [START_REF] Ueda | Enhanced pressor response to angiotensin I in normotensive men with the deletion genotype [DD] for angiotensin-converting enzyme[END_REF]. In turn, angiotensin II increases oxidative stress through NADH/NADPH oxidase stimulation and, in concert with other mediators such as cytokines, NO, endothelin-1, prostaglandins and Rho-protein pathway, promotes vascular inflammation, increases capillary permeability and impairs endothelial function [START_REF] Ruiz-Ortega | Renal and vascular hypertension-induced inflammation: role of angiotensin II[END_REF]. In that latter perspective, it was of interest the homogeneous forearm vasodilatory responsiveness to ACH, a negative result in agreement with previous reports [START_REF] Rossi | Exclusion of the ACE D/I gene polymorphism as a determinant of endothelial dysfunction[END_REF], suggestive of a different impact of the ACE genotypic variants on the capillary endothelial cell monolayer localized at the blood-vessel interface and endothelial cells covering resistance-sized forearm arterioles. Other possibilities cannot, however, be excluded including that, while TERalb measures systemic capillary permeability in basal conditions under the influence of several and interacting stimuli [START_REF] Hinsbergh | Endothelial permeability for macromolecules. Mechanistic aspects of pathophysiological modulation[END_REF][START_REF] Mehta | Signaling mechanisms regulating endothelial permeability[END_REF][START_REF] Parving | Microvascular permeability to plasma proteins in hypertension and diabetes mellitus in man-on the pathogenesis of hypertensive and diabetic microangiopathy[END_REF], the infused forearm model test only endogenous NO bio-availability in response to ACH, a NO-releasing stimulus [START_REF] Furchgott | The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine[END_REF]. Furthermore, several and heterogeneous organs and tissues contribute to the transvascular albumin leakage determination while forearm responses represent a single, mainly muscular vascular bed. Finally, NOmediated relaxation needs only endothelial synthesis and diffusion of the substance to underlying smooth muscle cells, while capillary permeability requires a more complicated functional and structural interaction of cell junctions and extra-cellular matrix with endothelial cells [START_REF] Hinsbergh | Endothelial permeability for macromolecules. Mechanistic aspects of pathophysiological modulation[END_REF][START_REF] Mehta | Signaling mechanisms regulating endothelial permeability[END_REF][START_REF] Parving | Microvascular permeability to plasma proteins in hypertension and diabetes mellitus in man-on the pathogenesis of hypertensive and diabetic microangiopathy[END_REF]. On the Dell'Omo et al: ACE polymorphism and capillary permeability in hypertension, 8/2/2006 CS2006/0165 other hand, the preserved forearm responsiveness to locally infused ACH, concordant with previous data in forearm [e.g. [START_REF] Cockcroft | Preserved endotheliumdependent vasodilatation in patients with essential hypertension[END_REF][START_REF] Bruning | In vivo characterization of muscarinic receptor subtypes that mediate vasodilatation in patients with essential hypertension[END_REF], coronary [e.g. 31] and subcutaneous [e.g. 32] arterioles, shows that impaired endothelial-mediated vasodilatation is not universal among essential hypertensive patients but may rather develop at more complicated stages [START_REF] Perticone | Prognostic significance of endothelial dysfunction in hypertensive patients[END_REF] or with longer duration of hypertensive disease [34]. However, we have no arguments in favor or against those possibilities, and, on the other hand, our study did not address those specific issues.

Comment also deserves the confirmed [1-5] association of higher UAE with the ACE D/D genotype, and more importantly in this context, its association with TERalb suggestive of some shared genetically-promoted determinant for the abnormal behavior of renal and systemic microvessels in ACE D/D hypertensive patients. Previous data showed that more albumin leaks through exaggeratedly permeant glomeruli characterized by decreased size-and charge-selectivity [35] and that albuminuria may reflect a generalized transvascular albumin leakiness in clinically healthy and diabetic subjects [36,37]. It should be noted, however, that the correlation between TERalb and UAE, albeit statistically significant, was rather weak in our present group. We also could not find a similar behavior in previous series [START_REF] Pedrinelli | Microalbuminuria and transvascular albumin leakage in essential hypertension[END_REF], perhaps because the modulation of both parameters by multiple hemodynamic, metabolic and behavioral factors [START_REF] Pedrinelli | Microalbuminuria, an integrated marker of cardiovascular risk in essential hypertension[END_REF][START_REF] Mehta | Signaling mechanisms regulating endothelial permeability[END_REF] may obscure that link. It may not be irrelevant to note that higher UAE predicts cardiovascular events even in non hypertensive individuals [38] and MA is frequent among patients with mildly elevated BP, lean body size and no evidence of metabolic abnormalities and subclinical inflammation [START_REF] Pedrinelli | Low-grade inflammation and microalbuminuria in hypertension[END_REF]. In those subsets, perhaps, genetic influences may be more influential on the renal and systemic microvascular system, an attractive possibility to be tested in the future.

Limitations of the study

A first obvious limitation of our study regards the strength of conclusions based upon a sample size of 79 patients. However, calculations accounting for effect size and allele frequency for predisposing alleles [START_REF] Hattersley | What makes a good genetic association study?[END_REF] indicate that, albeit limited, those numbers have a high statistical power to detect significant association when, as in our conditions, homozygous susceptibility genotype has a large biological impact (allelic OR=3.1) and the prevalence of the susceptibility allele in the control group is high (41%). On the other hand, we are aware that small initial studies frequently overestimate the true effect size thus increasing sample size requirements to thousands of cases and controls [e.g. 41], a goal difficult to be achieved dealing with a laborious parameter such as TERalb. For this reason, our present work should be seen as a biologically plausible, hypothesisgenerating contribution waiting for support from larger trials. Second, we studied an all-male hypertensive group and the influence of the ACE I/D polymorphism may differ by gender [START_REF] O'donnell | Evidence for association and genetic linkage of the angiotensin-converting enzyme locus with hypertension and blood pressure in men but not women in the Framingham Heart Study[END_REF].

  0002) or stratified by ACE genotype (D/D: 7.0±2.0 %/hr, n=6 vs 10.4±2.7 %/hr, n=36, p=0.005; I/D+I/I: 7.8±1.1 %/hr, n=10 vs 9.3±2.1, n=43, p=0.03). In the hypertensive sample, TERalb was higher (p=0.03) in D/D homozygotes than I/D+I/I subjects (figure 1) independent of age, BP levels, metabolic parameters, renal function and MetS frequency that matched closely in the two hypertensive subgroups (table 2). Hematocrit (I/D+I/I: 38.3±4.7% vs D/D: 39.5±8.3%), serum albumin (I/D+I/I: 4.3±0.3 vs D/D: 4.6±0.3 g/dl) and plasma volume (I/D+I/I: 2837.3±359.4 vs D/D: 2900.5±400.5 ml/1.73 m 2 ) did not differ.When TERalb (as a dichotomized variable, cutoff: 11.1%/hr) was used as the dependent variable in a multivariate logistic regression model, the allelic odds ratio for ACE D/D vs pooled D/I+II was 3.1 (95% CI:1.1-7.5, p=0.04) after accounting for BP, LDL cholesterol, smoking status, presence of MetS, HOMA index.

  UAE and FBF responses by ACE genotype UAE (12.1 [16) vs 8.0(7.3), p=0.03) was higher and MA (42% vs 14%, p=0.01) more frequent in D/D hypertensive homozygotes in whom TERalb correlated positively with UAE (r=0.23, p=0.03, n=79) but not office and 24hr BP, BMI, lipids, fasting and post-load plasma glucose and insulin, HOMA index. No correlation was found among normotensive controls (figure 2). Baseline FBF (I/D+I/I: 3.7±1.3 , n=35 vs D/D: 3.6±1.1 ml 100 ml -1 forearm volume min -1 , n=31) and FBF responses to intra-brachial ACH and SNP did not differ by ACE genotype nor by hyper-vs normotensive status (figure 3) DISCUSSION The major and original finding of this cross-sectional genetic association study in never treated, uncomplicated hypertensive men was the independent association between ACE D/D homozygosis and faster TERalb. The data is consistent with a more deranged endothelial barrier function at the level of the systemic capillary circuit where most of the albumin permeation process takes place [7-9 for reviews]. Faster TERalb also coexisted with higher UAE suggesting some common genetically-mediated determinant for the abnormal behavior of renal and systemic Dell'Omo et al: ACE polymorphism and capillary permeability in hypertension, 8/2/2006 CS2006/0165 microvessels without evidence of NO-mediated endothelial dysfunction of systemic arterioles as assessed by forearm ACH stimulation.

Clinical Science Immediate Publication. Published on 4 Aug 2006 as manuscript CS20060165 Copyright 2006 The Biochemical Society Dell'Omo et al: ACE polymorphism and capillary permeability in hypertension, 8/2/2006 CS2006/0165

  5, 1, 1.5 and 2 hours after administration of the glucose load. Plasma glucose was measured by the glucooxidase method

	using a Beckman Glucose Analyzer I/I (Beckman Instruments), and plasma insulin by
	immunoradiometric assay (Biosource, no crossreactivity with human proinsulin) with an interassay
	variation coefficient of 5%.

Science Immediate Publication. Published on 4 Aug 2006 as manuscript CS20060165 Copyright 2006 The Biochemical Society Dell'Omo et al: ACE polymorphism and capillary permeability in hypertension, 8/2/2006 CS2006/0165 sensitivity

  1 [12-HR URINE VOLUME X URINE CREATININE]/SERUM CREATININE X 1.73/BODY SURFACE AREA . BMI was calculated as weight/height 2 (Kg/m 2 ). Metabolic syndrome (MetS) was diagnosed according to the National Cholesterol Education Program (NCEP) Adult Treatment Panel (ATP) III

2 FASTING SERUM INSULIN [μU/ML] X FASTING PLASMA GLUCOSE [MMOL/L]/22.5 6 Clinical

Clinical Science Immediate Publication. Published on 4 Aug 2006 as manuscript CS20060165 Copyright 2006 The Biochemical Society Dell'Omo et al: ACE polymorphism and capillary permeability in hypertension, 8/2/2006 CS2006/0165

	of their
	low number making separate statistical comparisons not meaningful, I/I subjects were grouped with
	I/D in statistical analyses.

Clinical Science Immediate Publication. Published on 4 Aug 2006 as manuscript CS20060165 Copyright 2006 The Biochemical Society FIGURES Figure 1 :

  Third, our sample was recruited cross-sectionally making it difficult to assess cause-effect TERalb by ACE genotype in hypertensive men. Data reported as Box-and-whisker plots3 . * p=0.03. For further details see text.

	TERalb (%) TERalb (%/	8.3 10.3 12.3 14.3 16.3	*
		6.3
		ID+II	DD
		N=43	N=36

hr) Clinical Science Immediate Publication. Published on 4 Aug 2006 as manuscript CS20060165 Copyright 2006 The Biochemical Society Figure 2:

  The relationship between TERalb and urinary albumin excretion [UAE, log-scale] in hypertensive patients [n=79, ]. For a comparison, the scatterplot also show data obtained in control subjects [n=16, •]. For statistics see text.

	TABLES
		1000
	UAE (ug/min)	10 100
		1
		4	6	8	10	12	14	16
				TERalb (%/hr)

Table 1 :

 1 Comparison of hypertensive patients and normotensive controls [Means±SD or geometric means [interquartile range]]. For statistics, definitions and abbreviations see text.

	Clinical
	VARIABLES	CONTROLS	HYPERTENSIVES	P VALUE
		N=16	N=79
	Age [yrs]	47±12	48±10	NS
	DD/ID/II	6/8/2	36/36/7	NS
	Clear creat [ml/min*1.73 m 2 ]	104±29	110±36	NS
	Active smokers	7/16 [43%]	26/79 [33%]	NS
	Office systolic BP [mm Hg]	126±10	149±14	N/A
	Office diastolic BP [mm Hg]	76±6	96±11	N/A
	24 hr systolic BP [mmHg]	121±7	137±16	N/A
	24-hr diastolic BP [mmHg]	75±6	88±9	N/A
	Fasting plasma glucose [mg/dL]	88±12	98±11	p=0.002
	Glucose AUC 0-120min [[mg/dl x 2h -1 ]x10 -3 ] 14.3±2.1	16.1±3.1	p=0.02
	Fasting insulin [μU/ml]	7.7 [2.3]	12.3 [10.4]	p=0.004
	Insulin AUC 0-120min [[μU/ml x 2h -1 ]x10 -3 ] 5.3 [6.1]	12.3 [10.4]	p=0.014
	HOMA-IR [Units]	1.7 [0.9]	2.9 [2.8]	p=0.0013
	HDL cholesterol [mg/dL]	46±9	45±14	NS
	Serum triglycerides [mg/dL]	94 [42]	160 [124]	p=0.004
	BMI [Kg/m 2 ]	24.1±1.7	27.3±3	p=0.0001
	MetS	0/16 [0%]	32/79 [41%]	p=0.005
	Total cholesterol [mg/dL]	193±47	208±42	NS
	LDL cholesterol [mg/dL]	127±43	126±38	NS

Science Immediate Publication. Published on 4 Aug 2006 as manuscript CS20060165 Copyright 2006 The Biochemical Society Dell'Omo et al: ACE polymorphism and capillary permeability in hypertension, 8/2/2006 CS2006/0165Table 2 :

 2 Comparisons by ACE genotype in hypertensive patients [Means±SD or geometric means [interquartile range]]. For definitions and abbreviations see text. None of the parameters showed significant differences.

	VARIABLES	ID +I/I	D/D
		N=43	N=36
	Age [yrs]	47±9	49±11
	D Allele frequency [%]	42%	100%
	Clear creat [ml/min*1.73 m 2 ]	106 [35]	110 [28]
	Active smokers	16/43 [37%]	10/36 [28%]
	Office systolic BP [mm Hg]	149±14	151±16
	Office diastolic BP [mm Hg]	96±11	96±9
	24 hr systolic BP [mmHg]	138±17	136±14
	24-hr diastolic BP [mmHg]	88±10	88±8
	Fasting plasma glucose [mg/dL]	99±11	96±11
	Glucose AUC 0-120min [[mg/dl x 2h -1 ]x10 -3 ] 16.7±2.7	15.1±3.2
	Fasting insulin [μU/ml]	13.9 [2.5]	10.4 [7]
	Insulin AUC 0-120min [[μU/ml x 2h -1 ]x10 -3 ] 13.9 [7.4]	7.4 [8.2]
	HOMA-IR [Units]	3.3 [3.3]	2.6 [1.7]
	HDL cholesterol [mg/dL]	45±15	44±12
	Serum triglycerides [mg/dL]	159 [147]	151 [113]
	BMI [Kg/m 2 ]	27.3±3	27.4±3.1
	MetS	20/43 [47%]	12/36 [33%]
	Total cholesterol [mg/dL]	208±42	213±37
	LDL cholesterol [mg/dL]	126±38	134±32

Box-and-Whisker plot:The central box encloses the middle 50 percent of the data ; the horizontal line inside the box represents the median and the mean is plotted as a cross. Vertical lines [whiskers] extend from each end of the box and cover four interquartile ranges.


Professor Christopher J Mathias
email: c.mathias@imperial.ac.uk

Dphil Dsc

Frcp Fmedsci

Beta adrenergic receptor blockers and the treatment of vasovagal syncope -more nails in the coffin!

β-adrenergic receptor blockers are one of a number of therapeutic agents promoted as having beneficial effects in vasovagal syncope. In this issue of Clinical Science, Eldadah and coworkers have investigated the effect of the β-adrenergic receptor blocker propranolol in preventing syncope in a double-blind cross-over trail in eight subjects with a diagnosis based on tilt table testing and elevated plasma adrenaline levels during syncope. Of these, seven did not respond and the authors therefore suggest that this drug has no role in the management of vasovagal syncope. Their laboratory-based study, however, raises a number of issues regarding terminology, choice of subjects, the value and role of investigations directed towards diagnosis and in understanding pathophysiological mechanisms and the relevance of such trial to individual subjects with vasovagal syncope.

Beta-adrenergic receptor blockers are one of a number of therapeutic agents promoted as having beneficial effects in vasovagal syncope. This is a common condition that affects all age groups. It also is known as 'common' or 'emotionally-induced' fainting. It falls under the umbrella of neurally-mediated syncope (also referred to as reflex syncope), which is a condition characterised by intermittent dysfunction of the autonomic nervous system [START_REF] Mathias | To stand on one's own legs[END_REF] . This causes withdrawal of sympathetic tone resulting in vasodilatation and thus hypotension, and an increase in parasympathetic (vagal) activity resulting in bradycardia. Vasodepression, cardioinhibition or the combination, reduce cerebral blood flow thus causing syncope, especially in the upright position. The prognosis in vasovagal syncope is favourable even if untreated, unlike certain other causes of neurally-mediated syncope, and especially in syncope caused by cardiac dysrhythmias. However, in vasovagal syncope the uncertainty of attacks, the potential for trauma, possible difficulties with driving, and limitations in pursuing certain jobs, all contribute to concerns and morbidity.

A number of stimuli often are recognised as provoking an episode of vasovagal syncope [START_REF] Ganzeboom | Prevalence and triggers for syncope in medical students[END_REF] . These include fear of needles, blood and pain in addition to situations predisposing to a fall in blood pressure when upright, such as intense heat, impaired fluid repletion and excessive fluid loss. The precise mechanisms resulting in vasovagal syncope, whether central, peripheral or both, often are unclear. Various humoral agents, and in particular those that cause vasodilatation have been postulated as being causative or contributory. These include adrenaline, which in some studies is elevated during a syncopal episode. This has provided the theoretical basis for the use of beta-adrenergic receptor blockers in vasovagal syncope. Thus, it seems likely that blockade of vasodilatatory beta-2 receptors (with a non-selective blocker such as propranolol), will be more effective than cardioselective beta-1 blockers.

Copyright 2006 The Biochemical Society

In this issue the effect of propranolol in preventing syncope is reported in a double-blind crossover trial in 8 subjects with a laboratory confirmed diagnosis based on tilt table testing and elevated plasma adrenaline levels during syncope [START_REF] Eldadah | Failure of propranolol to prevent tilt-evoked system vasodilatation, adrenaline release and neurocardiogenic syncope[END_REF] . Of these 7 did not respond and the authors therefore suggest that this drug has no role in their management. Their study however raises a number of issues in vasovagal syncope regarding terminology, choice of subjects, the value and role of investigations directed towards diagnosis and in understanding pathophysiological mechanisms, and the relevance of such trials to individual patients.

Clarity in defining the disorder, and in the nomenclature used, is necessary. Neurally mediated syncope has a major advantage as it indicates a neurogenic cause through the autonomic nervous system that affects blood vessels, the heart or both, thus resulting in syncope [START_REF] Mathias | Orthostatic hypotension and orthostatic intolerance[END_REF] . It encompasses a number of disorders, including the most common, vasovagal syncope that may present at any age, often in the teenage years. In infants and young children, reflex anoxic syncope [START_REF] Stephenson | Paroxysmal non-epileptic disorders: differential diagnosis of epilepsy[END_REF] , which has both similarities and differences to vasovagal syncope needs to be considered, while in the elderly, carotid sinus hypersensitivity increasingly is recognised [START_REF] Mcintosh | Clinical characteristics of vasodepressor, cardioinhibitory and mixed carotid sinus syndrome in the elderly[END_REF] . Miscellaneous causes include micturition syncope, and rarer disorders such as associated with glossopharyngeal neuralgia and tumours [START_REF] Worth | Syncope associated with pain as the presenting feature of neck malignancy: failure of cardiac pacemaker to prevent attacks in two cases[END_REF] . In the study by Eldadah et al. [START_REF] Eldadah | Failure of propranolol to prevent tilt-evoked system vasodilatation, adrenaline release and neurocardiogenic syncope[END_REF] , the term 'neurocardiogenic' was used, synonymously with vasovagal syncope. This is misleading, as it implies neural mechanisms affecting the heart only. There are three sub-types of vasovagal syncope; cardio-inhibitory (with vagal activation), vasodepressor (with vasodilatation secondary to sympathetic withdrawal) and combined (mixed). The combined form is the most common. Identification of the sub-type is important as the management varies; in the cardioinhibitory form a cardiac pacemaker is more likely to be of benefit.

Clinical Science Immediate Publication. Published on 18 Jul 2006 as manuscript CS20060174

Copyright 2006 The Biochemical Society Investigations in vasovagal syncope focus on confirming the diagnosis, determining the sub-type and ideally understanding the pathophysiological mechanisms [START_REF] Mathias | Role of autonomic evaluation in the diagnosis and management of syncope[END_REF] . Tilt table testing is a key investigation. Alone it may not provoke an episode in the laboratory. In vasovagal syncope induced by needles or blood, head-up tilt in combination with venepuncture (or even pseudo puncture) [START_REF] Mathias | Observations on recurrent syncope and presyncope in 641 patients[END_REF] often induces an episode. Whether laboratory mimickry provides a measure of what occurs in real life remains unclear but often is the basis of diagnosis and therapeutic strategies.

Head-up tilt even with needle provocation may not induce a vasovagal episode, especially when nausea or pain is the key initiating stimulus. Some therefore advocate supraphysiological measures (such as lower body negative pressure), or a pharmacological stimulus (often a vasodilator), to induce an episode. This, however, raises questions about the relevance of laboratory-based testing to the real life clinical situation. There also is a further issue about reproducibility of tilt table testing. Recent studies in subjects with an implanted loop recorder indicate differences with tilt table and drug challenge studies; with the loop recorder however, a major deficiency is lack of blood pressure data [START_REF] Deharo | An implantable loop recorder study of highly symptomatic vasovagal patients: The heart rate rhythm observed during a spontaneous Clinical Science Immediate Publication. Published on 18 Jul 2006 as manuscript CS20060174 Copyright 2006 The Biochemical Society syncope is identical to the recurrent syncope but not correlated with the head-up tilt test or adenosine triphosphate test[END_REF] . Syncope sometimes may occur without a fall either in blood pressure or heart rate, as in Patient 4 [START_REF] Eldadah | Failure of propranolol to prevent tilt-evoked system vasodilatation, adrenaline release and neurocardiogenic syncope[END_REF] . Some argue that this may result from cerebral vasoconstriction alone; hyperventilation may be a contributing factor in this response. Alternatively, it may indicate an 'apparent' loss of consciousness, as in pseudosyncope. This diagnosis is extremely difficult to confirm and may co-exist with vasovagal syncope, which complicates matters further [START_REF] Mathias | Familial vasovagal syncope and pseudosyncope: observations in a case with both natural and adopted siblings[END_REF] .

The effect of the trial drug on associated disorders that contribute to the condition needs Copyright 2006 The Biochemical Society event [START_REF] Hajdinjak | Diagnosing postural tachycardia syndrome -head-up tilt or standing?[END_REF] . Some PoTS subjects respond to beta adrenergic receptor blockers, presumably by reducing tachycardia which precedes the vasovagal episode. Syncope also is more frequent in migraine sufferers [START_REF] Thijs | Syncope in migraine[END_REF] . Beta blockers, including propranolol, may reduce or prevent migrainous headaches [START_REF] Mathias | Orthostatic hypotension and orthostatic intolerance[END_REF] , and thus may reduce the frequency of syncopal episodes, consistent with clinical experience.

This study [START_REF] Eldadah | Failure of propranolol to prevent tilt-evoked system vasodilatation, adrenaline release and neurocardiogenic syncope[END_REF] also emphasises the possible errors that may result from meta-analysis. The studies included in such analyses may vary in the type of drug, the dosage, inclusion and exclusion criteria, and techniques to monitor benefit. There are likely to be difficulties especially in a heterogenous disorder such as vasovagal syncope. A previous meta-analysis [START_REF] Dendi | Meta-analysis of non-selective versus beta-1 adrenoceptorselective blockade in prevention of tilt-induced neurocardiogenic syndrome[END_REF] suggested that non-selective beta blockers were more likely to be effective than cardioselective blockers, but this was not confirmed in the detailed study with propranolol [START_REF] Eldadah | Failure of propranolol to prevent tilt-evoked system vasodilatation, adrenaline release and neurocardiogenic syncope[END_REF] . Two other recent studies also indicate that neither cardio-selective nor non-selective beta blockers are effective [START_REF] Flevari | Vasovagal syncope: a prospective, randomized, crossover evaluation of the effect of propranolol, nadolol and placebo on syncope recurrence and patients' wellbeing[END_REF][START_REF] Sheldon | Prevention of Syncope Trial (POST): A randomized, placebo-controlled study of metoprolol in the prevention of vasovagal syncope[END_REF] .

In conclusion therefore, this study has demonstrated clearly that the non-selective beta adrenergic receptor blocker propranolol was ineffective in preventing vasovagal syncope in a small group of subjects. It indirectly has raised a number of issues concerning diagnosis, investigation and management of this extremely common but complex disorder.

  consideration. One patient (Patient 4) had a substantial tachycardia during head-up tilt, of over 30 bpm, consistent with the postural tachycardia syndrome (PoTS). A recent study indicates that approximately 25% of PoTS subjects have vasovagal syncope confirmed with a laboratory Clinical Science Immediate Publication. Published on 18 Jul 2006 as manuscript CS20060174

Clinical Science Immediate Publication. Published on 18 Jul 2006 as manuscript CS20060174

Clinical Science Immediate Publication. Published on 18 Jul 2006 as manuscript CS20060174 Copyright 2006 The Biochemical Society


Zhen Huang

Leif Jansson

Åke Sjöholm
email: ake.sjoholm@sodersjukhuset.se

Vasoactive drugs enhance pancreatic islet

Keywords: Pancreatic islet, diabetes, blood flow, gender, pravastatin, captopril, irbesartan enzyme-linked immunosorbent assay, IBF, islet blood flow, IPGTT, intraperitoneal glucose tolerance test, KBF, kidney blood flow, NO, nitric oxide, PBF, pancreatic blood flow, RAS, renin-angiotensin system

Introduction

Type 2 diabetes is increasing in the Western world and is seen in ever-younger age groups.

The disease is characterized not only by hyperglycemia, but also insulin resistance with attendant dyslipidemia, hypertension and endothelial dysfunction. These aberrations may result in cardiovascular and renal disease, and adversely impact the prognosis of diabetic patients in terms of longevity and quality of life. Gender differences also exist, with more pronounced negative effects of diabetes on the lipid profile and blood pressure in women compared with men [START_REF] Howard | Adverse effects of diabetes on multiple cardiovascular disease risk factors in women. The Strong Heart Study[END_REF][START_REF] Walden | Sex differences in the effect of diabetes mellitus on lipoprotein triglyceride and cholesterol concentrations[END_REF]. The diabetic state may also impede the cardioprotective actions normally conferred by estrogen [START_REF] Steinberg | Type II diabetes abrogates sex differences in endothelial function in premenopausal women[END_REF]. To improve prognosis of diabetic patients, several risk factors other than glycemia (e.g. hypertension, albuminuria, dyslipidemia etc) need to be treated as well. Diabetic patients whose risk factor profile is well controlled are thus commonly being treated with one or more antidiabetic drugs, a lipid lowering statin and an ACE inhibitor or angiotensin receptor antagonist against hypertension and albuminuria.

The systemic renin-angiotensin system (RAS) plays a crucial role in the regulation of arterial blood pressure. In the past few years, it has become increasingly clear that local RAS also exist in various tissues, implying that high local levels of angiotensin II (Ang II) might exert paracrine influences on neighboring cells [START_REF] Leung | Pancreatic islet renin angiotensin system: its novel roles in islet function and in diabetes mellitus[END_REF]. In the pancreas of several species, mRNA encoding angiotensinogen and renin, as well as substantial levels of angiotensin II, have been detected [START_REF] Leung | Pancreatic islet renin angiotensin system: its novel roles in islet function and in diabetes mellitus[END_REF]. Ang II has been shown to adversely influence pancreatic and islet blood flow through vasocontractive effects [START_REF] Leung | Pancreatic islet renin angiotensin system: its novel roles in islet function and in diabetes mellitus[END_REF].

Interestingly, both ACE inhibitors or angiotensin receptor antagonists and certain statins have been reported to decrease the risk of developing diabetes in large clinical trials [START_REF] Sjoholm | Inflammation and the etiology of type 2 diabetes[END_REF]. However, the mechanisms behind these antidiabetic effects remain elusive. We recently showed stimulatory effects of these drugs on pancreatic islet blood flow in male rats [START_REF] Huang | Pancreatic islet blood flow is selectively enhanced by captopril, irbesartan and pravastatin, and suppressed by palmitate[END_REF]. In this paper, we aimed at evaluating the influence of ACE inhibition, angiotensin receptor antagonism and pravastatin treatment on pancreatic and islet blood flow, as well as on glycemia and insulin concentrations in normal female rats. Copyright 2006 The Biochemical Society Female Wistar rats (ScanBur, Sollentuna, Sweden), weighing 300 to 350 g, were used in all experiments. The animals had free access to pelleted food (Type R34; ScanBur, Sollentuna, Sweden) and tap water at all times. All experiments were approved by the local animal ethics committee at Uppsala University. Captopril and pravastatin were graciously donated by Bristol-Myers Squibb Company (New York, NY). Irbesartan was generously given by Sanofi-Synthelabo (Paris, France).

Materials and methods

Animals and drugs

Blood flow measurements

The experiments were performed according to a protocol previously described in detail [START_REF] Jansson | Stimulation by glucose of the blood flow to the pancreatic islets of the rat[END_REF].

The animals were anesthetized with an intraperitoneal injection of thiobutabarbital sodium (120 mg/kg body weight; Inactin™, Research Biochemicals International, Natick, MA), and placed on a heated operating table to maintain body temperature. Polyethylene catheters were inserted into the ascending aorta, via the right common carotid artery, and into the left femoral artery. The catheter in the aorta was connected to a pressure transducer (model PDCR 75/1, Druck Ltd., Groby, Leicestershire, UK) to allow constant monitoring of the mean arterial blood pressure. After the blood pressure was stable, the animals were injected intravenously with 1 ml of saline, 1 ml of pravastatin (0.5 mg/kg), 1 ml of irbesartan (3 mg/kg) or 1 ml of captopril (3 mg/kg). All these substances were dissolved in saline. Ten minuets later, 1.5-2.0 x 10 5 non-radioactive microspheres (IMT, Stason Labs., Irvine, CA), with a mean diameter of 10 μm, were injected during 10 s via the catheter with its tip located in the ascending aorta.

An arterial blood sample was collected from the catheter in the femoral arterial 5 s before the microsphere injection, and this process continued for a total of 60 s.

The exact withdrawal rate in each experiment was determined by weighing the sample.

Additional arterial blood samples were obtained and later analyzed for hematocrit, blood glucose, and serum insulin concentrations (see below). After the animals were killed by cervical dislocation, the whole pancreas and both adrenal glands, as well as a 100-mg slice of the left kidney (including both cortex and medulla) were collected. The microsphere contents in these organs were determined separately. The organs were treated with a freeze-thawing technique [START_REF] Jansson | A rapid method of visualizing the pancreatic islets for studies of islet capillary blood flow using non-radioactive microspheres[END_REF], which enabled the visualization and localization of the microspheres from either the endocrine or the exocrine parenchyma of the pancreas. This was achieved by applying a microscope (Zeiss MB6; Leica AB, Stockholm, Sweden) equipped with both bright and dark field illumination. The former type of illumination allowed us to count the microspheres, whereas the latter enabled localization of the microspheres from either the endocrine or Copyright 2006 The Biochemical Society exocrine parenchyma [START_REF] Jansson | A rapid method of visualizing the pancreatic islets for studies of islet capillary blood flow using non-radioactive microspheres[END_REF]. The number of microspheres in the islets and exocrine tissue was counted as previously described in detail [START_REF] Jansson | A rapid method of visualizing the pancreatic islets for studies of islet capillary blood flow using non-radioactive microspheres[END_REF]. The microsphere contents of the adrenal glands were used as a control to confirm an even distribution of the microspheres in the arterial circulation. The microsphere content of each of the arterial reference samples was determined by transferring the samples to glass microfiber filters and counting the microspheres in a

Intraperitoneal glucose tolerance test

An intraperitoneal glucose tolerance test (IPGTT) was performed simultaneously as the blood flow measurements. The animals were injected intraperitoneally with a 30 % (wt/vol) Dglucose solution (2 g glucose/kg body wt). Blood samples were drawn from the tail vein immediately before and 10, 30, 60, and 120 min after glucose administration. Area under the curve for the IPGTT was determined by computerized image analysis.

Measurement of glucose and insulin concentrations

Blood glucose concentrations were measured with test reagent strips (Medisense, Solna, Sweden) based on the glucose oxidase method and serum insulin concentrations were monitored with ELISA kit (Rat Insulin ELISA, Mercodia, Uppsala, Sweden). The insulin ELISA showed 98 % recovery upon addition. Coefficients of variation were 3.4 % within assay, 2.2 % between assays, and 3.9 % in total assay.

Statistical analysis

All

Results

Effects of captopril, irbesartan and pravastatin on blood flow in female rats. Intravenous injection of pravastatin (0.5 mg/kg BW), captopril (3 mg/kg BW) and irbesartan (3 mg/kg BW) significantly enhanced PBF (Figure 1a), the action of the statin being the most pronounced. IBF was also significantly augmented by all three substances (Figure 1b). Only irbesartan and captopril preferentially increased IBF, i.e., relatively more than PBF (IBF as % of PBF; Figure 1c). Renal blood flow was also substantially increased after administration of all three agents (Figure 2a). Irbesartan and pravastatin augmented adrenal blood flow significantly, whereas captopril did not (Figure 2b). Interestingly and quite unexpectedly, the vascular response to pravastatin was more pronounced than that occurring after giving agents interfering in RAS in all organs tested. Additionally, this effect occurred exclusively in female rats and not in our previously studied male rats. This indicates significant vasoactive actions of pravastatin that have hitherto not received much attention but that may be involved in the anti-atherogenic effects of the statin. This finding may also have implications for genderspecific differences in the vasoactive responses to these drugs described previously [START_REF] Fischer | Renin angiotensin system and gender differences in the cardiovascular system[END_REF][START_REF] Hayward | The roles of gender, the menopause and hormone replacement on cardiovascular function[END_REF].

Blood glucose concentrations, serum insulin levels, and mean arterial blood pressure.

None of the treatments influenced non-stimulated serum insulin levels significantly (Figure 3a). Blood glucose concentrations also did not differ between any of the treatment groups in the non-stimulated state (Figure 3b). In contrast, during an IPGTT late phase insulin secretion was substantially augmented (Figure 3a) and glucose tolerance was significantly improved (Figure 3b) in animals treated with captopril or pravastatin. Separate calculations of the total amount of insulin secreted during the 120 min IPGTT (area under the curve) revealed that insulin secretion in rats treated with pravastatin (p<0.01) or captopril (p<0.001) were significantly higher than in control rats receiving solvent only (Figure 3c). As shown in Figure 3d, there was also a trend towards a decrease in post-load glycemia, expressed as AUC, although this difference did not attain statistical significance. No effects on mean arterial

Discussion

Type 2 diabetes is increasing in the Western world and is seen in ever-younger age groups [START_REF] Zimmet | Global and societal implications of the diabetes epidemic[END_REF]. We can expect this to lead to momentous public health problems, especially in the form of premature cardiovascular morbidity. In terms of quantity, the most important complications of type 2 diabetes are macroangiopathies, i.e. myocardial infarction and stroke, which cause some 70 % of the deaths related to type 2 diabetes [START_REF] Gaede | Multifactorial intervention and cardiovascular disease in patients with type 2 diabetes[END_REF][START_REF] Zimmet | Global and societal implications of the diabetes epidemic[END_REF]. In contrast to microangiopathies (e.g. nephropathy and retinopathy), where the causal relation to hyperglycemia is well supported, the link between hyperglycemia and macroangiopathy is uncertain, at least in terms of the possibility of reducing macrovascular morbidity solely by reducing hyperglycemia [START_REF] Gaede | Multifactorial intervention and cardiovascular disease in patients with type 2 diabetes[END_REF]. To improve prognosis of diabetic patients, several other risk factors (e.g. hypertension, albuminuria [a biomarker of generalized endothelial dysfunction], dyslipidemia etc) need to be treated as well [START_REF] Gaede | Multifactorial intervention and cardiovascular disease in patients with type 2 diabetes[END_REF]. The disease is characterized not only by hyperglycemia, but also insulin resistance with attendant dyslipidemia and increased levels of circulating free fatty acids. Diabetic patients whose risk factor profile is well controlled are thus being treated with one or more antidiabetic drugs, a lipid lowering statin and an ACE inhibitor or angiotensin receptor antagonist against hypertension and albuminuria. These drugs are also exerting beneficial metabolic effects, causing an improved glucose tolerance in patients, but the precise nature of the mechanisms by which this is achieved remains elusive [START_REF] Sjoholm | Inflammation and the etiology of type 2 diabetes[END_REF]. We have now studied whether these drugs influence islet blood perfusion, glycemia and insulin levels in vivo in female rats.

Our present study shows a substantial increase in pancreatic blood flow, pancreatic islet blood flow, renal and adrenal blood flow by pravastatin treatment. Along with these beneficial effects, preferentially increasing islet blood flow, insulin secretion was augmented (preferentially late phase secretion) and glycemia was improved following an IPGTT. Future work, involving diabetic animals, will reveal whether stimulation of islet blood flow by similar means is sufficient to reverse experimental hyperglycemia. The antithrombotic and anti-inflammatory effects by pravastatin may play a role in enhancing endothelium-dependent vasodilation [START_REF] Auer | Clinical significance of pleiotropic effects of statins: lipid reduction and beyond[END_REF], lending support to our present findings of increased blood flow after pravastatin administration. Conversely, dyslipidemia -a salient feature in type 2 diabetes -is Copyright 2006 The Biochemical Society known to impair endothelium-mediated vasodilatation [START_REF] Leung | Beneficial effect of cholesterol-lowering therapy on coronary endothelium-dependent relaxation in hypercholesterolaemic patients[END_REF] and impaired endothelial function has been shown to result in diminished capillary recruitment [START_REF] Serne | Microvascular function relates to insulin sensitivity and blood pressure in normal subjects[END_REF]. Pravastatin has known beneficial effects on endothelial function [START_REF] Egashira | Reduction in serum cholesterol with pravastatin improves endothelium-dependent coronary vasomotion in patients with hypercholesterolemia[END_REF][START_REF] Muramatsu | Hemodynamic changes associated with reduction in total cholesterol by treatment with the HMG-CoA reductase inhibitor pravastatin[END_REF][START_REF] Williams | Pravastatin has cholesterollowering independent effects on the artery wall of atherosclerotic monkeys[END_REF]; therefore, pravastatin may significantly enhance selective tissue perfusion by restoring endothelial function. Beyond simply lowering cholesterol, it also exerts beneficial antithrombotic actions by inhibiting platelet aggregation and promoting local nitric oxide (NO) synthesis [START_REF] Sotiriou | Beneficial effects of statins in coronary artery disease--beyond lowering cholesterol[END_REF][START_REF] Svensson | Inhibition of nitric oxide synthase by NG-nitro-L-arginine causes a preferential decrease in pancreatic islet blood flow in normal rats and spontaneously diabetic GK rats[END_REF][START_REF] Tsuda | Effects of pravastatin sodium and simvastatin on plasma fibrinogen level and blood rheology in type II hyperlipoproteinemia[END_REF]. Thus, it is possible -albeit not proven -that increased local NO formation might be involved in the salutary effects of pravastatin noted in the current study. There appear to be important differences between different statins in this regard, as pravastatin is in a class of its own in terms of preventing diabetes [START_REF] Freeman | Pravastatin and the development of diabetes mellitus: evidence for a protective treatment effect in the West of Scotland Coronary Prevention Study[END_REF][START_REF] Sjoholm | Endothelial inflammation in insulin resistance[END_REF][START_REF] Sjoholm | Inflammation and the etiology of type 2 diabetes[END_REF]. Pravastatin is not metabolized by hepatic CYP-450 enzymes, shows very little binding to proteins, and is markedly hydrophilic. Whether these characteristics or other attributes, such as anti-inflammatory actions, underlie pravastatin's anti-diabetogenic effect remains to be shown. Other mechanisms are conceivable, for instance direct effects on the endocrine pancreas. In vitro studies have shown that lipophilic statins (simvastatin) inhibit glucose-stimulated insulin secretion by blocking voltage-gated L-type Ca 2+ channels in insulin-secreting β cells, whereas pravastatin has no such adverse effect [START_REF] Yada | Inhibition by simvastatin, but not pravastatin, of glucose-induced cytosolic Ca2+ signalling and insulin secretion due to blockade of L-type Ca2+ channels in rat islet beta-cells[END_REF]. Furthermore, pravastatin can prevent inflammation and rejection of transplanted islets of Langerhans [START_REF] Arita | Prevention of primary nonfunction of canine islet autografts by treatment with pravastatin[END_REF], all actions that would contribute to improved glucose tolerance.

We recently reported that administration of pravastatin in male Wistar rats increased only pancreatic islet blood flow [START_REF] Huang | Pancreatic islet blood flow is selectively enhanced by captopril, irbesartan and pravastatin, and suppressed by palmitate[END_REF]. Our present results indicate qualitative and quantitative gender-specific differences between male and female rats in terms of vascular responses to pravastatin and RAS interfering agents, which may be due to the vasoactive features of estrogen or other influences conferred by female gender. For instance, while in male rats captopril was the most potent stimulus of islet blood flow [START_REF] Huang | Pancreatic islet blood flow is selectively enhanced by captopril, irbesartan and pravastatin, and suppressed by palmitate[END_REF], pravastatin evoked the strongest stimulation of islet blood flow in the presently studied female rats (Fig 1B). Likewise, captopril was a stronger stimulator of islet blood flow in male than in female rats, compared to irbesartan. Additionally, non-stimulated serum insulin levels were heightened by irbesartan and captopril in male rats [START_REF] Huang | Pancreatic islet blood flow is selectively enhanced by captopril, irbesartan and pravastatin, and suppressed by palmitate[END_REF] but not influenced by these drugs in the present work on female rats. It is becoming increasingly appreciated that gender differences exist in terms of susceptibility to and mortality from a variety of vascular diseases [START_REF] Dubey | Cardiovascular protective effects of 17beta-estradiol metabolites[END_REF][START_REF] Hayward | The roles of gender, the menopause and hormone replacement on cardiovascular function[END_REF][START_REF] Mendelsohn | The protective effects of estrogen on the cardiovascular system[END_REF]. Estrogen is known to increase NO production primarily through upregulation of endothelial NO synthase (eNOS) gene expression [START_REF] Kauser | Potential cellular signaling mechanisms mediating upregulation of endothelial nitric oxide production by estrogen[END_REF]; also a gender difference exists in basal NO release by rabbit aorta [START_REF] Hayashi | Basal release of nitric oxide from aortic rings is greater in female rabbits than in male rabbits: implications for atherosclerosis[END_REF][START_REF] Hayashi | Gender differences in atherosclerosis: possible role of nitric oxide[END_REF]. Alternatively, it is possible that estrogen may function by upregulating eNOS Copyright 2006 The Biochemical Society and/or enhancing production of eNOS-derived NO [START_REF] Best | The effect of estrogen replacement therapy on plasma nitric oxide and endothelin-1 levels in postmenopausal women[END_REF][START_REF] Guetta | The role of nitric oxide in coronary vascular effects of estrogen in postmenopausal women[END_REF][START_REF] Rubanyi | Vascular estrogen receptors and endothelium-derived nitric oxide production in the mouse aorta. Gender difference effect of estrogen receptor gene disruption[END_REF], thereby producing a vasodilatory effect [START_REF] Mendelsohn | The protective effects of estrogen on the cardiovascular system[END_REF].

The systemic RAS plays a crucial role in the regulation of arterial blood pressure. In the past few years, it has become increasingly clear that local RAS also exist in various tissues, implying that high local levels of angiotensin II (Ang II) might exert paracrine influences on neighboring cells [START_REF] Carlsson | The renin-angiotensin system in the endocrine pancreas[END_REF][START_REF] Lau | Evidence for a local angiotensin-generating system and dose-dependent inhibition of glucose-stimulated insulin release by angiotensin II in isolated pancreatic islets[END_REF][START_REF] Leung | Tissue renin-angiotensin system: its expression, localization, regulation and potential role in the pancreas[END_REF][START_REF] Tahmasebi | The tissue renin-angiotensin system in human pancreas[END_REF][START_REF] Tikellis | Improved islet morphology after blockade of the renin-angiotensin system in the ZDF rat[END_REF]. In the pancreas of several species, mRNA encoding angiotensinogen and renin, as well as substantial levels of angiotensin II, have been detected [START_REF] Carlsson | The renin-angiotensin system in the endocrine pancreas[END_REF][START_REF] Lau | Evidence for a local angiotensin-generating system and dose-dependent inhibition of glucose-stimulated insulin release by angiotensin II in isolated pancreatic islets[END_REF][START_REF] Leung | Tissue renin-angiotensin system: its expression, localization, regulation and potential role in the pancreas[END_REF][START_REF] Tahmasebi | The tissue renin-angiotensin system in human pancreas[END_REF][START_REF] Tikellis | Improved islet morphology after blockade of the renin-angiotensin system in the ZDF rat[END_REF]. Ang II has been shown to adversely influence pancreatic and islet blood flow through vasocontractive effects [START_REF] Carlsson | Angiotensin II and the endocrine pancreas: effects on islet blood flow and insulin secretion in rats[END_REF][START_REF] Olsson | Local blood flow regulation in transplanted rat pancreatic islets: influence of adenosine, angiotensin II, and nitric oxide inhibition[END_REF]. Also, high affinity binding sites for Ang II were recently localized specifically to islet β-cells by double immunostaining and Ang II was found to block glucose-stimulated insulin secretion, an event fully reversible by losartan [START_REF] Carlsson | Angiotensin II and the endocrine pancreas: effects on islet blood flow and insulin secretion in rats[END_REF][START_REF] Lau | Evidence for a local angiotensin-generating system and dose-dependent inhibition of glucose-stimulated insulin release by angiotensin II in isolated pancreatic islets[END_REF][START_REF] Olsson | Local blood flow regulation in transplanted rat pancreatic islets: influence of adenosine, angiotensin II, and nitric oxide inhibition[END_REF]. It is thus conceivable that pancreatic Ang II, locally produced by intrinsic RAS, may adversely influence insulin secretion in vivo, either directly by suppressing β-cell insulin exocytosis or indirectly through inhibitory effects on islet blood perfusion [START_REF] Carlsson | Angiotensin II and the endocrine pancreas: effects on islet blood flow and insulin secretion in rats[END_REF][START_REF] Lau | Evidence for a local angiotensin-generating system and dose-dependent inhibition of glucose-stimulated insulin release by angiotensin II in isolated pancreatic islets[END_REF][START_REF] Olsson | Local blood flow regulation in transplanted rat pancreatic islets: influence of adenosine, angiotensin II, and nitric oxide inhibition[END_REF]. This may be of particular importance in diabetic patients since hypertension is markedly overrepresented in these individuals [START_REF] Sjoholm | Endothelial inflammation in insulin resistance[END_REF][START_REF] Sjoholm | Inflammation and the etiology of type 2 diabetes[END_REF], and angiotensinogen expression seems to be upregulated by hypertension [START_REF] Sjoholm | Endothelial inflammation in insulin resistance[END_REF][START_REF] Sjoholm | Inflammation and the etiology of type 2 diabetes[END_REF]. Hence, many diabetic patients are treated with ACE inhibitors or angiotensin receptor antagonists against their hypertension or as part of a renal protection strategy. About 15 years ago, it became clear that estrogen can influence the RAS [START_REF] Brosnihan | Effects of chronic hormone replacement on the renin-angiotensin system in cynomolgus monkeys[END_REF][START_REF] Hassager | The long-term effect of oral and percutaneous estradiol on plasma renin substrate and blood pressure[END_REF][START_REF] Laragh | Renin, angiotensin, aldosterone and hormonal regulation of arterial pressure and salt balance[END_REF][START_REF] Oelkers | Effects of estrogens and progestogens on the renin-aldosterone system and blood pressure[END_REF][START_REF] Sealey | Estradiol-and progesterone-related increases in the renin-aldosterone system: studies during ovarian stimulation and early pregnancy[END_REF][START_REF] Skinner | Alteration by oral contraceptives of normal menstrual changes in plasma renin activity, concentration and substrate[END_REF][START_REF] Weinberger | The effect of posture and saline loading on plasma renin activity and aldosterone concentration in pregnant, non-pregnant and estrogen-treated women[END_REF], by down-regulating renin, ACE, and Ang II receptor expression [START_REF] Usui | Regulation of angiotensin II receptor expression by nitric oxide in rat adrenal gland[END_REF]. An interesting review has reported that gender and sex hormones affect the components of the RAS by multifarious mechanisms [START_REF] Fischer | Renin angiotensin system and gender differences in the cardiovascular system[END_REF]. In general, estrogen inhibits Ang II action and has been shown to attenuate vascular contractility [START_REF] Rosenfeld | Estrogen-induced refractoriness to the pressor effects of infused angiotensin II[END_REF]. Irbesartan is an Ang II receptor antagonist, characterized by high selectivity and insurmountable blockade of the type 1 Ang II receptor. In our study, intravenous injection of irbesartan or the ACE inhibitor captopril induced robust increases in blood flow not only in the pancreas and pancreatic islets, but also (as expected) in the kidney.

Pancreatic blood flow was preferentially diverted to the endocrine part, i.e. the islets. This can be explained by a local RAS operative in the pancreas [START_REF] Leung | Pancreatic islet renin angiotensin system: its novel roles in islet function and in diabetes mellitus[END_REF]. Such a system enables local production of Ang II, yielding Ang II concentrations much higher than those encountered in peripheral blood. Some publications show that islet blood flow can be suppressed by locally produced Ang II [START_REF] Gaede | Multifactorial intervention and cardiovascular disease in patients with type 2 diabetes[END_REF][START_REF] Lau | Evidence for a local angiotensin-generating system and dose-dependent inhibition of glucose-stimulated insulin release by angiotensin II in isolated pancreatic islets[END_REF], lending support to our present findings. Recent evidence also suggests that certain Ang II receptor antagonists have the capacity to enhance vascular vasodilatation [START_REF] Gaede | Multifactorial intervention and cardiovascular disease in patients with type 2 diabetes[END_REF][START_REF] Lau | Evidence for a local angiotensin-generating system and dose-dependent inhibition of glucose-stimulated insulin release by angiotensin II in isolated pancreatic islets[END_REF], which might be another mechanism contributing to our current findings. Additionally, it was recently demonstrated that the Ang II receptor antagonist Copyright 2006 The Biochemical Society losartan selectively improved glucose-induced insulin release and insulin biosynthesis in islets from spontaneously diabetic mice [START_REF] Chu | Angiotensin II type 1 receptor blockade improves beta-cell function and glucose tolerance in a mouse model of type 2 diabetes[END_REF]. Oral losartan treatment delayed the onset of diabetes, and improved glucose tolerance in the diabetic mice, but did not affect the insulin sensitivity of peripheral tissues, suggesting that Ang II receptor antagonism improves β-cell function and glycemia in young type 2 diabetic mice. It was also recently shown that another potent Ang II receptor antagonist, candesartan, when given orally over several weeks increased β-cell mass, increased staining intensity of insulin, and decreased oxidative stress markers in β-cells [START_REF] Shao | Beneficial effects of candesartan, an angiotensin II type 1 receptor blocker, on beta-cell function and morphology in db/db mice[END_REF].

Candesartan treatment also reduced fibrosis in and around the islets and prevented the loss of endothelial cells in islets, indicating that candesartan partially prevents deterioration of glucose tolerance by providing protection against progressive β-cell damage in diabetic mice.

In conclusion, we have shown that vasoactive drugs that are frequently given to diabetic patients and improve their glucose tolerance have beneficial effects by enhancing pancreatic islet blood flow, augmenting insulin secretion and improving glycemia. Our findings moreover indicate qualitative gender differences in the vascular response to these drugs, which might be related to the ability of estrogen to produce vasodilatation. The results may prove useful in tailoring gender-specific treatment strategies that improve islet function, contributing to an improved glycemic control in diabetic patients or in subjects at risk of developing glucose intolerance.

Legends to figures

  stereomicroscope. The blood flow rates were calculated according to the formula Q org = Q ref x N org /N ref , where Q org denotes organ blood flow (mL/min), Q ref denotes withdrawal rate of the reference sample (mL/min), N org denotes number of microspheres in the organ and N ref denotes the number of microspheres in the reference sample.

  values are given as means ± SEM for the number of experiments given. Each experiment represents one animal and experiments were performed on different days. Statistical comparisons were made with one-way analysis of variance (ANOVA) (SigmaStat; SSPD, Clinical Science Immediate Publication. Published on 5 Oct 2006 as manuscript CS20060176 Copyright 2006 The Biochemical Society Erfart, Germany) in conjunction with Bonferronni's post-hoc test.. P < 0.05 was deemed statistically significant.

Clinical

  Science Immediate Publication. Published on 5 Oct 2006 as manuscript CS20060176 Copyright 2006 The Biochemical Society blood pressure (averaging 110 mmHg) were detected by any of the treatments given (data not shown).

Figure 1 :

 1 Figure 1: Enhancement of pancreatic blood flow by captopril, irbesartan, and pravastatin. Following i.v. injection of captopril (3 mg/kg BW), irbesartan (3 mg/kg BW), or pravastatin (0.5 mg/kg BW) into normal female rats, rates of blood perfusion in the whole pancreas (a), pancreatic islets (b), and the fraction of total pancreatic blood flow contributed by islets (c) were measured using a microsphere technique. Bars represent means ± SEM for 8 independent experiments. *, ** and *** denote p<0.05, p<0.01 and p<0.001, respectively, for chance differences vs controls using ANOVA.

Figure 2 :

 2 Figure 2: Enhancement of renal blood flow and adrenal blood flow by captopril, irbesartan, and pravastatin. Following i.v. injection of captopril (3 mg/kg BW), irbesartan (3 mg/kg BW), or pravastatin (0.5 mg/kg BW) into normal female rats, rates of blood perfusion in the kidneys (a) and adrenals (b) were measured using a microsphere technique. Bars represent means ± SEM for 8 independent experiments. * and ** denote p<0.05 and p<0.01, respectively, for chance differences vs controls using ANOVA.

Figure 3 :

 3 Figure 3: Captopril and pravastatin.augment insulin secretion and improve glycemia during an IPGTT. Following i.v. injection of captopril (3 mg/kg BW) or pravastatin (0.5 mg/kg BW) into normal female rats, serum insulin concentrations (a) were measured with ELISA and blood glucose levels (b) with test reagent strips during a simultaneous IPGTT over 120 min. Changes in insulin (c) and glucose (d) responses are expressed as area under the curve integrated over 120 min. Values represent means ± SEM for 6 independent experiments. *, ** and *** denote p<0.05, p<0.01 and p<0.001, respectively, for chance differences vs controls using ANOVA.

  Figure 1a

  Figure 1c

Clinical Science Immediate Publication. Published on 5 Oct 2006 as manuscript CS20060176

Clinical Science Immediate Publication. Published on 5 Oct 2006 as manuscript CS20060176

Clinical Science Immediate Publication. Published on 5 Oct 2006 as manuscript CS20060176

Clinical Science Immediate Publication. Published on 5 Oct 2006 as manuscript CS20060176

Clinical Science Immediate Publication. Published on 5 Oct 2006 as manuscript CS20060176

Science Immediate Publication. Published on 5 Oct 2006 as manuscript CS20060176 Copyright 2006 The Biochemical Society

  Barndiabetesfonden, Åke Wiberg's Foundation, Torsten and Ragnar Söderberg's Foundations, Berth von Kantzow's Foundation, Harald Jeansson's and Harald and Greta Jeansson's Foundations, Tore Nilson's Foundation for Medical Research, Fredrik and Inger Thuring's Foundation, and Syskonen Svensson's Fund.

	Clinical

Acknowledgments

The skilled technical assistance of Birgitta Bodin and Astrid Nordin is gratefully acknowledged. Financial support was provided through the regional agreement on medical training and clinical research (ALF) between Stockholm county council and the Karolinska Institute and also financially supported by the Swedish Research Council (72X-109, 72X-12550, 72X-14507, 72X-11564, 72P-12995, 72X-00034, 72X-09890, 72XS-12708, 72P-14787, and 12P-10151), EFSD/Novo Nordisk for Type 2 Diabetes Research Grant, the Swedish Diabetes Association, the NOVO Nordic Research Fund, the Family Ernfors Fund, Petrus and Augusta Hedlund's Foundation, the Swedish Society of Medicine, Trygg-Hansa's Research Foundation, the Janne Elgqvist Family Foundation, the Sigurd and Elsa Golje Memorial Foundation, Svenska Försäkringsföreningen, Svenska Diabetesstiftelsen, Magn. Bergvall Foundation, Novo-Nordisk Sweden Pharma AB, Stiftelsen Samariten,