Zhen Huang

Leif Jansson

Åke Sjöholm
email: ake.sjoholm@sodersjukhuset.se

Gender-specific regulation of pancreatic islet blood flow, insulin levels, and glycemia in spontaneously diabetic Goto-Kakizaki rats

Keywords: Pancreatic islet, diabetes, blood flow, gender, pravastatin, captopril, candesartan, irbesartan and palmitate islet blood flow, KBF, kidney blood flow, NO, nitric oxide, PBF, pancreatic blood flow, RAS, renin-angiotensin system

Diabetic patients are often treated with a statin against hyperlipidemia and an ACE inhibitor or angiotensin receptor antagonist against hypertension or albuminuria.

These drugs may also exert beneficial metabolic effects, causing an improved glucose tolerance in patients. Gender-related differences have also been observed in the clinical responsiveness to these drugs, but the mechanism behind this is unclear. We now studied whether these drugs and the fatty acid palmitate influence pancreatic microcirculation, thereby impacting insulin secretion and glycemia in vivo, in spontaneously diabetic male and female Goto-Kakizaki rats. In male rats, islet blood flow (IBF) and total pancreatic blood flow (PBF) were increased significantly by pravastatin, captopril and irbesartan. Serum insulin levels were increased by pravastatin and captopril. Palmitate suppressed IBF and raised blood glucose. In female animals, IBF was stimulated by captopril, candesartan and irbesartan. PBF was increased by captopril, candesartan and irbesartan, and by pravastatin. Palmitate suppressed IBF and serum insulin secretion. In conclusion, the present study lends support to the view that a local pancreatic RAS and pravastatin may be selectively influencing pancreatic microcirculation and therefore affecting insulin secretion and glycemia. Free fatty acids impaired islet blood flow, suppressed insulin secretion, and raised blood glucose. Substantial gender-related differences in the vascular and metabolic responses to these drugs prevail in this diabetic animal model.

Introduction

The renin-angiotensin system (RAS) has been extensively studied since the first identification of renin by Tigerstedt and Bergmann in 1898 [START_REF] Paul | Physiology of local renin-angiotensin systems[END_REF]. It plays an important role in regulating blood volume and systemic vascular resistance [START_REF] Peach | Renin-angiotensin system: biochemistry and mechanisms of action[END_REF]. It has become increasingly clear that a local RAS exists in the pancreas and some other tissues [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] Tahmasebi | The tissue renin-angiotensin system in human pancreas[END_REF][START_REF] Tikellis | Improved islet morphology after blockade of the reninangiotensin system in the ZDF rat[END_REF].

Ang II, considered the main effective peptide of the RAS, has been shown to adversely influence pancreatic and islet blood flow through vasocontractive effects thereby suppressing insulin secretion [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]. ACE, another component of RAS, has been detected in endothelial cells of the vascular wall [START_REF] Corvol | Recent advances in knowledge of the structure and function of the angiotensin I converting enzyme[END_REF][START_REF] Costerousse | Angiotensin I-converting enzyme (kininase II) in cardiovascular and renal regulations and diseases[END_REF][START_REF] Falkenhahn | Cellular distribution of angiotensin-converting enzyme after myocardial infarction[END_REF] in human and rat [START_REF] Hirsch | Tissue-specific activation of cardiac angiotensin converting enzyme in experimental heart failure[END_REF][START_REF] Kreutz | Induction of cardiac angiotensin I-converting enzyme with dietary NaCl-loading in genetically hypertensive and normotensive rats[END_REF][START_REF] Paul | Gene expression of the renin-angiotensin system in human tissues. Quantitative analysis by the polymerase chain reaction[END_REF][START_REF] Schunkert | Increased rat cardiac angiotensin converting enzyme activity and mRNA expression in pressure overload left ventricular hypertrophy. Effects on coronary resistance, contractility, and relaxation[END_REF]. This may be particular importance in diabetic patients since many of them are treated with ACE inhibitors or angiotensin receptor antagonists against hypertension or as part of a renal protection effort. Another salient feature of diabetic patients is hyperlipidemia and elevated serum levels of free fatty acids; therefore many diabetic patients use lipid lowering drugs, most notable statins. Interestingly, gender differences were observed in large clinical trials, 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], and also the vascular responses to statins and RAS-interfering drugs seem to differ between men and women [START_REF] Dale | Impact of gender on statin efficacy[END_REF][START_REF] Juilliere | Response to drug therapy of cardiac failure according to gender[END_REF]. Recently, we have shown the stimulatory effects of such drugs on pancreatic islet blood flow in normal male [START_REF] Huang | Pancreatic islet blood flow is selectively enhanced by captopril, irbesartan and pravastatin, and suppressed by palmitate[END_REF] and female [START_REF] Huang | Vasoactive drugs enhance pancreatic islet blood flow, augment insulin secretion and improve glucose tolerance in female rats[END_REF] Wistar rats. However, it is not known how these drugs influence islet microcirculation, insulin secretion and glucose tolerance in diabetic animals. In the present study, we thus investigated gender-specific effects of ACE inhibition, Ang II receptor antagonism, statin treatment, and palmitate administration on islet blood flow, insulin levels and glycemia in Goto-Kakizaki rats, a widely used animal model of human type 2 diabetes [START_REF] Portha | Programmed disorders of beta-cell development and function as one cause for type 2 diabetes? The GK rat paradigm[END_REF].

Materials and Methods

Animals and drugs

Male (300-350 g) and female (200-250 g) Goto-Kakizaki rats were obtained from our KISÖS breeding colony (Animal Department, Stockholm South Hospital, Stockholm, Sweden). The Stockholm GK rat colony was established at KISÖS in 2004 from breeding pairs graciously donated by Prof. Robert V. Farese (Tampa, FL). All animals had free access to pelleted food and tap water and were kept under standard conditions. The experiments were approved by the local ethical committee.

Pravastatin and captopril were kindly given by Bristol Myers Squibb (New York, NY).

Irbesartan was generously given by Sanofi-Aventis (Paris, France), whereas candesartan was graciously donated by Astra Zeneca (Södertälje, Sweden). Sodium palmitate was bought from Sigma-Aldrich (St Louis, MO) and dissolved in 10 % ethanol solution.

Surgical preparation

The rats were anesthetized with an intraperitoneal injection of sodium thiobutabarbital (120 mg/kg of body weight; Inactin™; Research Biochemicals International [Natick, MA]), then placed on a heated operating table in order to maintain body temperature.

Catheters were inserted into the ascending aorta via the right 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) in order to constantly monitor the mean arterial blood pressure. After the mean arterial blood pressure had remained stable for at least 20 min, the animals were injected intravenously with 1 ml of saline, 1 ml of pravastatin (0.5 mg/kg of body weight), 1 ml of captopril (3 mg/kg of body weight), 1 ml of candesartan (10 mg/kg of body weight), or 1 ml of irbesartan (3 mg/kg of body weight). The irbesartan was tested separately due to delayed delivery. All substances were dissolved in saline.

The palmitate experiments (60 mg/ml) were done in a separate series because the fatty acid had to be dissolved in 10 % ethanol, and thus controls were given equal amounts of this solvent only. To this end, 1 ml palmitate or solvent was injected i.v. exactly as described for the other agents above.

Blood flow measurements

The procedure was performed according to a protocol described in detail previously [START_REF] Jansson | Stimulation by glucose of the blood flow to the pancreatic islets of the rat[END_REF]. Briefly, 10 minutes after the injection of test substances, about 1.5-2.0 x 10 5 non-radioactive microspheres (IMT, Stason Labs, Irvine, CA), with a mean diameter of 10 μm, were injected into the ascending aorta during 10 s. Starting 5 s before microsphere injection and continuing for a total 60 s, an arterial blood reference sample was collected from the catheter in the femoral artery. The amount of blood was confirmed by weighting each reference sample. Additional arterial blood samples were collected at the end of the experiment (approximately 30 minutes after the surgical procedure has been done) for later measurement of blood glucose and serum insulin concentrations.

The animals were then 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 removed, blotted, and weighted. The microsphere contents of the adrenals, part of kidney, pancreatic islets, and exocrine parenchyma were determined in frozen-thawed preparations separately [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 blood flow values were calculated according to the formula: Q org = Q ref x N org /N ref , where Q org is organ blood flow (ml/min), Q ref is withdrawal rate of the reference sample (ml/min), N org is the number of microspheres in the organ and N ref is the number of microspheres in the reference sample. A less than 10 % difference in microsphere content between the two adrenal glands was taken to confirm an even distribution of the microspheres in the arterial blood stream.

Blood glucose and insulin concentration determinations

The blood glucose concentration was analyzed with test strips based on the glucose oxidase method (Medisense, Solna, Sweden), and serum insulin concentrations were measured with an ELISA kit (rat insulin ELISA; Mercodia, Uppsala, Sweden).

Statistical calculations

All values are given as the mean ± SEM. Probabilities of chance differences between experimental groups were analyzed with one-way ANOVA (SigmaStat) in combination with Student's two-tailed unpaired t test. P<0.05 was considered statistical significant.

Results

Gender-specific effects of captopril, candesartan, pravastatin, and irbesartan on blood flow

In male rats, captopril (3 mg/kg BW), pravastatin (0.5 mg/kg BW) and irbesartan (3 mg/kg BW) all enhanced PBF (Fig. 1a,1b) and IBF (Fig. 2a,2b) significantly, whereas candesartan had no discernable effects (Fig. 1a,2a). KBF was enhanced markedly by pravastatin (P<0.01), candesartan (P<0.05) and irbesartan (P<0.05), but not significantly by captopril (Table 1). Only pravastatin increased ABF significantly (P<0.05), whereas captopril, candesartan and irbesartan failed to do so (Table 1).

In female rats, all test substances augmented PBF markedly (Fig 1a ,1b).

Administration of captopril, candesartan and irbesartan induced an increase in IBF (Fig. 2a,2b), whereas pravastatin did not. KBF was markedly increased by pravastatin (P<0.001), captopril (P<0.001), candesartan (P<0.001) and irbesartan (P<0.01) (Table 1). Captopril and candesartan increased ABF significantly (both P<0.01), whereas pravastatin and irbesartan had no such effects (Table 1).

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

In male GK rats, pravastatin and captopril enhanced serum insulin levels (Table 2), whereas irbesartan and candesartan did not. The blood glucose concentration was unexpectedly increased by captopril (Table 2). There were no significant differences between any groups in serum insulin levels in the female animals (Table 3). Surprisingly, however, pravastatin and captopril slightly raised blood glucose, which might be due to surgical stress. Irbesartan had no effects on serum insulin or blood glucose levels (Table 3). No effects were found on mean arterial pressure by any of the treatments given (Table 2, 3).

Effects of palmitate on blood flow, insulin levels and glycemia

Palmitate significantly decreased islet blood flow in both male (Fig. 3) and female (Fig. 3) rats but had no significant changes on whole pancreatic blood flow in either gender (not shown). The reason for gender differences in basal islet blood flow (Figure 3) is unknown, but might be related to increased vascular sensitivity to ethanol in female rats (perhaps in part due to lower body weight than males). In male rats, palmitate markedly increased blood glucose (Table 2) along with a non-significant decrease in serum insulin (Table 2). In female rats, palmitate lowered serum insulin concentration significantly (Table 3) but failed to significantly impact glycemia in these animals (Table 3). In female rats only, palmitate evoked an increase in ABF but did not influence KBF in either gender (Table 1).

Discussion

Type 2 diabetes is increasing in prevalence globally and is seen in ever-younger age groups [START_REF] Zimmet | Global and societal implications of the diabetes epidemic[END_REF]. The disease is characterized not only by hyperglycemia, but also insulin resistance with attendant hypertension, dyslipidemia and increased levels of circulating free fatty acids. Most diabetic patients are being treated with one or more antidiabetic drugs, a lipid lowering statin, and an ACE inhibitor or angiotensin receptor antagonist against hypertension and/or albuminuria. In clinical trials, it has been repeatedly observed that these kinds of drugs also influence the rate of incident diabetes [START_REF] Abuissa | Angiotensin-converting enzyme inhibitors or angiotensin receptor blockers for prevention of type 2 diabetes: a meta-analysis of randomized clinical trials[END_REF], causing an improved glucose tolerance in patients. Consistent with this, we have previously shown that these drugs exert substantial short-term impact on islet blood perfusion, insulin secretion and glucose tolerance in vivo in normal Wistar rats of both male and female gender [START_REF] Huang | Pancreatic islet blood flow is selectively enhanced by captopril, irbesartan and pravastatin, and suppressed by palmitate[END_REF][START_REF] Huang | Vasoactive drugs enhance pancreatic islet blood flow, augment insulin secretion and improve glucose tolerance in female rats[END_REF]. To evaluate whether these drugs also influence islet microcirculation, insulin levels and glycemia under diabetic conditions, we have presently studied the effects of these drugs in spontaneously diabetic GK rats in both male and female groups.

Our findings reveal that irbesartan, an angiotensin II receptor antagonist, and captopril, an ACE inhibitor, induced a robust increase in islet and whole pancreatic blood flow both in male and female GK rats. This beneficial effect confirms our previous study with the administration of these drugs to normal Wistar rats of both genders [START_REF] Huang | Pancreatic islet blood flow is selectively enhanced by captopril, irbesartan and pravastatin, and suppressed by palmitate[END_REF][START_REF] Huang | Vasoactive drugs enhance pancreatic islet blood flow, augment insulin secretion and improve glucose tolerance in female rats[END_REF]. Captopril also significantly increased serum insulin concentrations in male GK rats. Previous publications show that locally produced Ang II suppressed islet blood flow [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. Additionally, recent evidence also suggests that irbesartan has the capacity to enhance 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.

Recently, another Ang II receptor antagonist, candesartan, proved efficacious in reducing fibrosis in and around the islets and prevented the loss of endothelial cells in diabetic mouse islets by long term treatment, suggesting that candesartan may partially prevent deterioration of glucose tolerance by affording protection against progressive β-cell damage [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][START_REF] Shao | Angiotensin II receptor blocker provides pancreatic beta-cell protection independent of blood pressure lowering in diabetic db/db mice[END_REF]. Our results reveal that candesartan increased islet blood flow and total pancreatic blood flow significantly only in female GK rats, but not in male animals. This gender difference was not noted for irbesartan or captopril.

The underlying mechanism behind this notion remains elusive at this point, but, gender-related differences in the responsiveness to RAS-interfering drugs have been long noticed. A large body of evidence has been obtained through research in animals that components of the RAS are significantly influenced by gender at the tissue levels [START_REF] Armando | Estrogen upregulates renal angiotensin II AT(2) receptors[END_REF][START_REF] Ellison | Androgen regulation of rat renal angiotensinogen messenger RNA expression[END_REF][START_REF] Freshour | Gender differences in cardiac ACE expression are normalized in androgen-deprived male mice[END_REF][START_REF] Gallagher | Estrogen regulation of angiotensin-converting enzyme mRNA[END_REF][START_REF] Gordon | Regulation of angiotensinogen gene expression by estrogen[END_REF][START_REF] Hinojosa-Laborde | Ovariectomy augments hypertension in aging female Dahl salt-sensitive rats[END_REF][START_REF] Nickenig | Estrogen modulates AT1 receptor gene expression in vitro and in vivo[END_REF][START_REF] Nickenig | Differential effects of estrogen and progesterone on AT(1) receptor gene expression in vascular smooth muscle cells[END_REF], while clinical studies in humans have predominantly evaluated gender effects on the circulating, i.e. systemic, RAS [START_REF] Chidambaram | Variation in the renin angiotensin system throughout the normal menstrual cycle[END_REF][START_REF] Danser | Determinants of interindividual variation of renin and prorenin concentrations: evidence for a sexual dimorphism of (pro)renin levels in humans[END_REF][START_REF] Goldhaber | Plasma renin substrate, renin activity, and aldosterone levels in a sample of oral contraceptive users from a community survey[END_REF][START_REF] Harvey | The effects of different types and doses of oestrogen replacement therapy on clinic and ambulatory blood pressure and the renin-angiotensin system in normotensive postmenopausal women[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 | Oral contraceptives. Renin, aldosterone, and high blood pressure[END_REF][START_REF] Schunkert | Effects of estrogen replacement therapy on the renin-angiotensin system in postmenopausal women[END_REF][START_REF] Seely | Effects of conjugated oestrogen and droloxifene on the renin-angiotensin system, blood pressure and renal blood flow in postmenopausal women[END_REF]. The characterization of an estrogen-responsive element in the 5'-flanking region of the angiotensinogen gene was an important early finding to prove an interaction with sex hormones and the RAS at the molecular level [START_REF] Feldmer | Glucocorticoid-and estrogen-responsive elements in the 5'-flanking region of the rat angiotensinogen gene[END_REF][START_REF] Gordon | Regulation of angiotensinogen gene expression by estrogen[END_REF]. More recent studies indicate that renin is suppressed by estrogen [START_REF] Schunkert | Effects of estrogen replacement therapy on the renin-angiotensin system in postmenopausal women[END_REF]. Collectively, these studies are in line with the observation that plasma renin levels are lower in women compared with men [START_REF] Fischer | Renin angiotensin system and gender differences in the cardiovascular system[END_REF][START_REF] Schunkert | Effects of estrogen replacement therapy on the renin-angiotensin system in postmenopausal women[END_REF].

In addition, AT1 receptors may also be down-regulated by estrogen while estrogen deficiency leads to up-regulation of this receptor [START_REF] Nickenig | Estrogen modulates AT1 receptor gene expression in vitro and in vivo[END_REF]. In female transgenic rats with activated RAS, it was shown that estrogen treatment may afford protection against hypertension, possibly by amplifying the vasodilator contributions of Ang-(1-7), while reducing the formation and vasoconstrictor actions of Ang II [START_REF] Brosnihan | Estrogen protects transgenic hypertensive rats by shifting the vasoconstrictor-vasodilator balance of RAS[END_REF]. Conceivably, any of these mechanisms (or other) could contribute to our findings of gender-related differences in this system.

Pravastatin, a HMG-CoA reductase inhibitor, is clinically used in order to reduce the cholesterol level and thereby prevent dyslipidemia. Our study shows a preferential increase in pancreatic islet blood flow and pancreatic blood flow, as well as a significantly increased serum insulin level, in male GK rats by pravastatin administration. In contrast, in female animals total pancreatic blood flow was enhanced but not islet blood flow. In our previous studies in female non-diabetic Wistar rats, pravastatin actually increased both panceatic islet blood flow and total pancreatic blood flow [START_REF] Huang | Vasoactive drugs enhance pancreatic islet blood flow, augment insulin secretion and improve glucose tolerance in female rats[END_REF]. The reason for this difference is currently unknown.

However, with increasing age, persistent hyperglycemia may induce islet hypoperfusion in GK rats [START_REF] Olsson | The pancreatic islet endothelial cell: emerging roles in islet function and disease[END_REF]. The islet blood hypoperfusion is accomanied by an islet capillary hypertension. Such shear stress changes are known to change the gene expression in surrounding cells [START_REF] Olsson | The pancreatic islet endothelial cell: emerging roles in islet function and disease[END_REF], possibly impacting islet function. Dyslipidemia, a salient feature in type 2 diabetes, is known to impair endothelial-mediated vasodilatation [START_REF] Leung | Beneficial effect of cholesterol-lowering therapy on coronary endothelium-dependent relaxation in hypercholesterolaemic patients[END_REF][START_REF] Serne | Microvascular function relates to insulin sensitivity and blood pressure in normal subjects[END_REF]. Pravastatin has beneficial anti-inflammatory effects on endothelial function [START_REF] Auer | Clinical significance of pleiotropic effects of statins: lipid reduction and beyond[END_REF][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 cholesterol-lowering independent effects on the artery wall of atherosclerotic monkeys[END_REF], and therefore may significantly influence selective tissue perfusion.

When making comparisons with the diabetes preventive actions of pravastatin and RAS-interfering drugs noted in clinical trials [START_REF] Sjoholm | Inflammation and the etiology of type 2 diabetes[END_REF], it should be kept in mind that our present results reflect only very acute effects of the substances tested. While reports on long-term effects of such drugs on islet blood flow are scarce [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][START_REF] Tikellis | Improved islet morphology after blockade of the reninangiotensin system in the ZDF rat[END_REF], this interesting issue is worthy of further investigation. However, it would be beyond the scope of the present study, whose aim was to monitor short-term actions. Type 2 diabetes is commonly associated with elevated serum free fatty acids, due to insulin resistance and increased lipolysis [START_REF] Freeman | Pravastatin and the development of diabetes mellitus: Control 9[END_REF][START_REF] Gaede | Multifactorial intervention and cardiovascular disease in patients with type 2 diabetes[END_REF][START_REF] Sjoholm | Inflammation and the etiology of type 2 diabetes[END_REF][START_REF] Zimmet | Global and societal implications of the diabetes epidemic[END_REF]. Free fatty acids are generally believed to be involved in endothelial dysfunction and vascular damage in type 2 diabetes [START_REF] Freeman | Pravastatin and the development of diabetes mellitus: Control 9[END_REF][START_REF] Gaede | Multifactorial intervention and cardiovascular disease in patients with type 2 diabetes[END_REF][START_REF] Sjoholm | Inflammation and the etiology of type 2 diabetes[END_REF][START_REF] Zimmet | Global and societal implications of the diabetes epidemic[END_REF]. Our previous study showed that palmitate preferentially suppressed pancreatic islet blood flow in non-diabetic male Wistar rats [START_REF] Huang | Pancreatic islet blood flow is selectively enhanced by captopril, irbesartan and pravastatin, and suppressed by palmitate[END_REF]. In the present study, palmitate decreased both whole pancreatic and islet blood flow in both male and female GK rats. This might be one mechanism by which free fatty acids may negatively impact β-cell function, i.e. through impeding nutritive islet blood flow and thereby further worsening the diabetic state by limiting the supply of insulin needed to curb hyperglycemia. Our results in this respect are at variance with many previous findings that acute effects of saturated fatty acids on glucose induced insulin secretion are normally positive (58), both in vitro and in vivo, and we have no obvious explanation for this apparent discrepancy that deserves further investigation in future work.

In summary, several vasoactive drugs that are frequently given to diabetic patients, and reportedly may improve their glucose tolerance, selectively enhanced pancreatic islet blood flow in this animal model of type 2 diabetes. Gender-related differences in the vascular responses to these drugs were also noted in this system. Free fatty acids may aggravate hyperglycemia in diabetes by limiting islet blood flow as described here.

Legends to figures

  evidence for a protective treatment effect in the West of Scotland Coronary Prevention Study. Circulation. 103, 357-62. 58. Newsholme, P., Keane, D., Welters, H.J. and Morgan, N.G. Life and death decisions of the pancreatic beta-cell: the role of fatty acids. (2007) Clin Sci (Lond). 112, 27-42.

Figure 1 :

 1 Figure 1: Enhancement of pancreatic blood flow by pravastatin, captopril, candesartan and irbesartan in male and female GK rats. Following i.v. injection of pravastatin (0.5 mg/kg BW), captopril (3 mg/kg BW) or candesartan (10 mg/kg BW) into male GK rats, rates of blood perfusion in the whole pancreas (1a) were measured using microsphere technique. Separate experiments with irbesartan (3 mg/kg BW) are shown in (1b). Bars represent means ± SEM for 5 independent experiments. * and ** denote P<0.05 and P<0.01, respectively, for chance differences vs controls using ANOVA.

Figure 2 :

 2 Figure 2: Enhancement of pancreatic islet blood flow by pravastatin, captopril, candesartan and irbesartan in male and female GK rats. Following i.v. injection of pravastatin (0.5 mg/kg BW), captopril (3 mg/kg BW) or candesartan (10 mg/kg BW) into male GK rats, rates of blood perfusion in pancreatic islets (2a) were measured using a microsphere technique. Separate experiments with irbesartan (3 mg/kg BW) are shown in (2b). Bars represent means ± SEM for 5 independent experiments. * and ** denote P<0.05 and P<0.01, respectively, for chance differences vs controls using ANOVA.

Figure 3 :

 3 Figure 3: Suppression of pancreatic islet blood flow by palmitate in both male and female GK rats.Following i.v. injection of palmitate (60 mg/kg BW) or ethanol (solvent) into male or female GK rats, pancreatic islet blood flow was measured using a microsphere technique. Bars represent means ± SEM for 5 independent experiments. * denotes P<0.05 for a chance difference vs controls using ANOVA.

  Figure 1

Table 2 .

 2 Blood glucose was measured with test strips based on the glucose oxidase method. Serum insulin concentrations were analyzed with an ELISA kit. Blood pressure was constantly monitored by a pressure transducer. Data are means ± SE. * P<0.05; ** P<0.01; *** P<0.001 when compared with the corresponding control strain (Student's unpaired t test and ANOVA).

	In male GK group	Blood Glucose	Serum Insulin	Blood Pressure
		(mmol/l)	(ng/ml)	(mmHg)
	Control	7.7 ± 0.4	4.46 ± 0.4	98.6 ± 2.7
	Pravastatin	7.6 ± 0.6	7.19 ± 1.2 *	101.6 ± 3.4
	Captopril	10.9 ± 1 **	8.89 ± 1.3 ***	108 ± 2.4
	Candesartan	9.4 ± 1.4	5.35 ± 0.8	107 ± 2.7
	Ethanol	8.2 ± 0.6	8.19 ± 1.9	110 ± 2.9
	Palmitate	12.6 ± 1.9 *	5.49 ± 0.9	115 ± 4.7
	Control	8.26 ± 0.3	4.08 ± 0.1	95.5 ±1.9
	Irbesartan	7.58 ± 0.6	4.22 ± 0.4	103.7 ± 3.7

Table 3 .

 3 Blood glucose was measured with test strips based on the glucose oxidase method. Serum insulin concentrations were analyzed with an ELISA kit. Blood pressure was constantly monitored by a pressure transducer. Data are means ± SE. * P<0.05; ** P<0.01; *** P<0.001 when compared with the corresponding control strain (Student's unpaired t test and ANOVA).

	In female GK group	Blood Glucose	Serum Insulin	Blood Pressure
		(mmol/l)	(ng/ml)	(mmHg)
	Control	8.3 ± 0.6	3.05 ± 0.25	94.2 ± 3.5
	Pravastatin	11.2 ± 0.4 ***	3.09 ± 0.26	98.1 ± 1.3
	Captopril	11.3 ± 0.6 ***	3.01 ± 0.09	102 ± 3.6
	Candesartan	9.7 ± 11	3.14 ± 0.12	105 ± 2.3
	Ethanol	9.3 ± 0.8	4.15 ± 0.43	109 ± 5.9
	Palmitate	9.95 ± 0.5	3.1 ± 0.17 *	110 ± 1.7
	Control	9.16 ± 0.4	3.08 ± 0.1	95.1 ±1
	Irbesartan	10.4 ± 0.6	3.1 ± 0.1	105.2 ± 6.1

S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 21 Jan 2008 as manuscript CS20070386 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070386 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 Clinical Science Immediate Publication. Published on 21 Jan 2008 as manuscript CS20070386 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070386 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Acknowledgments

The skilled technical assistance of Birgitta Bodin and Astrid Nordin is gratefully acknowledged. Financial support was received from Astra-Zeneca Sweden AB, 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 Swedish-American Diabetes Research Program funded by the Juvenile Diabetes Foundation and the Wallenberg Foundation, the NOVO Nordic Research Fund, the Family Ernfors Fund, the Swedish Society of Medicine, the Sigurd and Elsa Golje Memorial Foundation, Svenska Försäkringsföreningen, Svenska Diabetesstiftelsen, Magn. Bergvall Foundation, 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


Wibke Reinhard

Klaus Stark

Kamil Sedlacek

Marcus Fischer

Andrea Baessler

Katharina Neureuther

Stefan Weber

Bernhard Kaess

Silke Wiedmann

Stefan Mitsching

Wolfgang Lieb

Jeanette Erdmann

Christa Meisinger

Angela Doering

Ralf Tolle║

Andreas Jeron

Guenter Riegger

Christian Hengstenberg
email: christian.hengstenberg@klinik.uni-regensburg.de

PPAR-alpha Polymorphisms and Myocardial Infarction 1/21 Reinhard et al. Association between PPAR-alpha Gene Polymorphisms and Myocardial Infarction

Keywords:

Peroxisome proliferator activated receptor alpha (PPARα) regulates the expression of genes that are involved in lipid metabolism, tissue homeostasis, and inflammation.

Consistent rodent and human studies suggest a link between PPARα function and cardiovascular disease, qualifying PPARα as a candidate gene for coronary artery disease. We comprehensively evaluated common genetic variations within the PPARα gene and assessed their association with myocardial infarction.

First, we characterized the linkage disequilibrium within the PPARα gene in an initial case-control sample of 806 individuals from the Regensburg Myocardial Infarction Family Study using a panel of densely spaced single nucleotide polymorphisms (SNPs) across the gene. Single SNP analysis showed significant association with the disease phenotype (OR=0.74, P=0.012, 95% CI=0.61-0.94 for rs135551). Moreover, we identified a protective 3-marker haplotype with an association trend for myocardial infarction (OR=0.76, P=0.067, 95% CI=0.56-1.92). Subsequently, we were able to confirm the single SNP and haplotype association results in an independent second case-control cohort with 667 cases from the Regensburg Myocardial Infarction Family Study and 862 control individuals from the WHO MONICA Augsburg project (OR=0.87, P=0.046, 95% CI=0.72-0.99 for rs135551; OR=0.80, P=0.034, 95% CI=0.65-0.98 for the 3-marker haplotype, respectively).

From these cross-sectional association results, we provide evidence that common variations in the PPARα gene may influence the risk of myocardial infarction in a European population.

INTRODUCTION

Myocardial infarction (MI) is considered a complex disease. Its development and progression underlies interactions between environmental and genetic influences [START_REF] Marenberg | Genetic susceptibility to death from coronary heart disease in a study of twins[END_REF]. Genetic association studies of candidate genes focus on the identification of genetic variations that contribute to or protect against the development of the disease [START_REF] Cardon | Association study designs for complex diseases[END_REF].

Peroxisome proliferator activated receptor alpha (PPARα), a member of the nuclear receptor superfamily, qualifies as an interesting candidate gene for atherosclerotic disease. PPARα is a ligand-activated transcription factor that regulates the transcription of genes involved in lipid metabolism, tissue homeostasis and proteins that initiate an inflammatory response central to atherosclerotic plaque formation [START_REF] Fruchart | Peroxisome proliferator-activated receptor-alpha activators regulate genes governing lipoprotein metabolism, vascular inflammation and atherosclerosis[END_REF].

Ligands for PPARα include fibrates, a substance class of lipid lowering drugs that has been shown to reduce the risk of cardiovascular events [4;5] Previously, genetic examinations of the PPARα locus have mainly focussed on the L162V polymorphism, which is the only functional variant that has so far been shown to be associated with changes in serum concentrations of lipids and lipoproteins, and with left ventricular hypertrophy [START_REF] Flavell | Variation in the PPARalpha gene is associated with altered function in vitro and plasma lipid concentrations in Type II diabetic subjects[END_REF][START_REF] Jamshidi | Peroxisome proliferator--activated receptor alpha gene regulates left ventricular growth in response to exercise and hypertension[END_REF][START_REF] Tai | Association between the PPARA L162V polymorphism and plasma lipid levels: the Framingham Offspring Study[END_REF][START_REF] Vohl | Molecular scanning of the human PPARa gene: association of the L162V mutation with hyperapobetalipoproteinemia[END_REF]. Yet, association studies between the L162V variant and atherosclerosis are limited and provide inconsistent results [START_REF] Flavell | Peroxisome proliferator-activated receptor alpha gene variants influence progression of coronary atherosclerosis and risk of coronary artery disease[END_REF]. However, to date, no comprehensive evaluation of common variations within the PPARα gene and their association with MI was performed. Therefore, the aims of our study were [START_REF] Marenberg | Genetic susceptibility to death from coronary heart disease in a study of twins[END_REF] to assess the linkage disequilibrium (LD) structure of the PPARα gene and to identify possible haplotypes in an initial case-control sample, (2) to perform single SNP and haplotype analyses for association with MI, and (3) to validate available positive results in an independent second case-control sample. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

MATERIALS AND METHODS

Study population

All MI patients were participants of the Regensburg MI Family Study. Selection criteria have been described previously [11;12]. In brief, we identified MI families, in which the index patient suffered from MI at the age of 60 years or younger and at least one more family member had been affected with MI and/or severe coronary artery disease (CAD), e.g. percutaneous coronary intervention or coronary bypass surgery). The diagnosis of MI was established by review of medical records according to the MONICA (Monitoring Trends and Determinants in Cardiovascular Disease) diagnostic criteria (http://www.ktl.fi/publications/monica/manual/index.htm). All MI patients and all of their available siblings and spouses were contacted and invited to participate in the study. Medical history, risk factor evaluation, clinical examination with anthropometric measurements, and blood samples were obtained from all participants.

For the initial case-control study, 433 unrelated MI patients fulfilling our index criteria and 373 unrelated unaffected control subjects were randomly selected to establish Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Phenotyping

At recruitment, all patients were studied by comparable design as previously described including a standardized interview, clinical examination, and biochemical as well as molecular analyses either at a visit at a study center (patients from the MONICA Augsburg study) or at a home visit by a physician (patients from rehabilitation centers) [12;14;15]. Validation of cardiovascular events at study entry was performed by review of medical records. Resting blood pressure was taken according to MONICA guidelines after participants had been resting in a sitting position [START_REF] Mayer | Association between arterial pressure and coronary artery calcification[END_REF]. Body weight in kilogram and height in meters were determined with subjects wearing light clothing. Blood samples were taken from non-fasting individuals. Serum levels of low-and high-density lipoprotein cholesterol and triglycerides were measured by standard enzymatic methods [START_REF] Holmer | Lipoprotein lipase gene polymorphism, cholesterol subfractions and myocardial infarction in large samples of the general population[END_REF]. Characteristics of both cohorts are shown in Table 1. (4) compatibility with our genotyping platform. To allow to determine the extend of LD beyond the boundaries of the gene, one SNP each in the 5' intergenic region and in the 3' UTR was included.

SNPs and Genotyping Methods

Seventeen

Genomic DNA was extracted from EDTA blood samples by standard procedures.

High throughput genotyping was performed on the ABI 7900 platform according to the manufacturer's protocol using the ABI assay on-demand or by-design service.

Genotyping quality was ensured by repetition of 10% (n=233) of all genotypes in independent PCR reactions. Genotypes did not differ between duplicates. For five duplicates, the definite genotype call was obtained in one of the assays with an undetermined call in the other. The overall genotyping call-rate was >98% for all SNPs. In individuals from the second sample, only rs135557, rs135551, rs135543, and rs135539 were genotyped. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Statistical Analyses

For each of the 17 SNPs, we tested whether the observed genotype frequencies in MI cases and controls deviated from Hardy-Weinberg equilibrium (P = 0.05 level).

Single SNP association analyses were performed in both case-control samples.

Allele and genotype frequencies were compared using the χ² test and Armitage test for trend reporting odds ratios (OR) and 95% confidence intervals (CI). The Armitage's trend test under the assumption of an additive mode of inheritance allows the reduction of genotype comparisons to one degree of freedom [START_REF] Armitage | Test for linear trends in proportions and frequencies[END_REF].

Additionally, pair-wise LD was determined applying the standard definition of r² [START_REF] Ardlie | Patterns of linkage disequilibrium in the human genome[END_REF].

A haplotype block was defined as a region in which all pair-wise r² values were >0.4.

Haplotypes and their frequencies were inferred from phase-unknown genotype data using PHASE version 2.1 [20;21]. Uncertain haplotypes with a probability of correct phase or allele <90% were excluded from the analyses. The calculated haploblock A as well as the number of copies of hap2 were tested for disease association by a χ² test. For interaction analyses all pair-wise combinations of SNPs were tested in a population-based case/control setting implemented in plink v0.99s [START_REF] Purcell | PLINK: A Tool Set for Whole-Genome Association and Population-Based Linkage Analyses[END_REF]. A two-sided Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

P

RESULTS

PPARα linkage disequilibrium mapping and haplotype structure

We characterised the LD and haplotype structure of the PPARα gene by genotyping a case-control sample of 806 individuals with (n=433) and without (n=375) MI for 17

SNPs (Table 1). There were 14 intronic SNPs, one SNP was positioned in exon 6, and one SNP was located in the 5' intergenic region and the 3' UTR, respectively, Reconstruction of haploblock A revealed 3 common haplotypes with a frequency >0.10 which were termed hap1, hap 2, and hap3 with frequencies of 54.3%, 28.7% and 12.3%, respectively. They comprised 95.3% of total chromosomes of the screening sample population.

yielding

Association of PPARα variants with MI in the initial case-control cohort

Initially, we carried out single marker association tests with all 17 SNPs the first cohort was genotyped for calculating odds ratios (OR) from comparisons of allele and genotype frequencies in cases and controls (Table 2). We found a significant association between rs135551 and MI (P=0.012, OR=0.74, 95% CI=0.61-0.94;

Armitage's trend test). Additionally, an association trend between rs135543 and MI could be observed resulting from an overrepresentation of the rare alleles in control individuals (P=0.082, OR=0.81, 95% CI=0.68- As both SNPs are parts of haploblock A, we next performed SNP haplotype association analysis using the three most common haplotypes hap1 -hap3.

Haplotype analyses may provide more power to detect association with disease than single marker analyses alone [START_REF] Newton-Cheh | Genetic association studies of complex traits: design and analysis issues[END_REF]. In the initial sample, we found no statistical significant association between haplotypes hap1 -hap3 and MI (Table 3). However, hap2, which is defined by the combination of the rare alleles, showed a trend for association with MI (P=0.067, OR=0.76, 95% CI=0.56-1.02, None of the other SNP markers was significantly associated with MI. In particular, we did not find a relation between rs1800206, the only SNP resulting in an amino acid exchange (L162V), and MI (Table 2). We further performed comprehensive testing for epistatic interaction between any of the 17 SNPs and found a statistically significant interaction with an asymptotic p-value threshold below 0.01 between rs135539 and rs11704979 (P=0.001, data not shown). To avoid missing an effect that was not detected by single SNP association analyses, we investigated the other three haploblocks B, C and D for possible haplotype association. These analyses did not reveal significant association results (data not shown). For further association analyses, we therefore focussed on haplotype block A as well as on the single markers contributing to or surrounding it.

Confirmation of association between MI and PPARα variants in an independent second case-control sample

In order to corroborate the data, we sought to validate our results in an independent second case-control cohort. Therefore, additional 1,529 individuals (667 MI cases from the Regensburg MI Family Study, 862 controls from the WHO MONICA Augsburg project) were genotyped for all SNPs of the 3-marker haplotype and one 5' adjacent SNP. In this second cohort, we were able to confirm single SNP association result for rs135551 (P=0.046, OR=0.87, 95% CI=0.72-0.99, Armitage's trend test, Table 4). Moreover, for rs135543 as well as hap2, which both showed a trend for association in the initial sample, now significant association with MI could be observed resulting from an over-representation in control individuals (P=0.014, OR=0.84, 95% CI=0.71-0.96, and P=0.034, OR=0.80, 95% CI=0.65-0.98, respectively; Tables 3 and4). Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

DISCUSSION

We comprehensively evaluated the LD structure of common genetic variations within the PPARα gene and assessed their potential relation with MI. In a two-step approach, we initially used a case-control sample to investigate the LD structure of the gene and to construct common haplotypes as well as to explore the association between single SNPs and haplotypes and MI. We found significant association of a single SNP marker (rs135551) with MI and a trend for association of the adjacent SNP (rs135543). Moreover, we identified a common three marker haplotype (hap2), consisting of the rare alleles of rs135551, rs135543 and rs135539, that was significantly associated with the disease phenotype. Consecutively, we were able to confirm our results in an independent second case-control sample. Here, we detected significant association with MI for the same two single SNP markers, rs135551 and rs135543, and hap2. Hap2 is over-represented in control individuals, thus it suggests protective properties against MI with an OR of 0.80 (P=0.034, 95% CI=0.65-0.98).

The mechanisms by which hap2 or its single marker variants influence the atherosclerotic process remain unclear. PPARα is extensively expressed in vascular cells, such as endothelial and smooth muscle cells or monocytes/macrophages [START_REF] Marx | Peroxisome proliferator-activated receptors and atherogenesis: regulators of gene expression in vascular cells[END_REF].

PPARα activation prompts anti-inflammatory effects at multiple stages of the atherosclerotic pathway [START_REF] Marx | PPARalpha activators inhibit cytokine-induced vascular cell adhesion molecule-1 expression in human endothelial cells[END_REF][START_REF] Pasceri | Modulation of C-reactive proteinmediated monocyte chemoattractant protein-1 induction in human endothelial cells by antiatherosclerosis drugs[END_REF][START_REF] Staels | Activation of human aortic smooth-muscle cells is inhibited by PPARalpha but not by PPARgamma activators[END_REF]. Thus, polymorphisms in the PPARα gene may affect this sequence via a functional mutation resulting for example in a defective protein or altered trans-activation or -repression potentials.

However, the genetic variations found in this study to be associated with the disease phenotype are positioned in intron 2. They neither insert a potential alternative splice site (visual checking for GT-AG consensus sites) nor do they include a consensus sequence for binding of an established transcription factor (analyzed by TRANSFAC 7.0 Public) [START_REF] Matys | TRANSFAC: transcriptional regulation, from patterns to profiles[END_REF]. It is therefore unlikely that one of these SNPs itself represents the causal mutation. We assume that an untested proximate marker in LD with hap2 might be a functional variant. SNP markers of hap2 are part of a weak but distinct LD block covering the promoter region and exon 1 to intron 2 as shown by HapMap data. Marker rs135557, located upstream to transcription start site, is not associated with MI in our study. Hence, the postulated causal mutation could be located between the core promoter region and intron 2 of the PPARα gene, but is unlikely to be located within the neighbouring gene. A possible effect of such a functional variant could be altered gene expression, as seen with the exonic PPARα L162V missense mutation, where the V allele possesses higher transcriptional activation in vitro [6;32]. In our study, the L162V variant did not show any association with MI. Other studies examining the impact of the L162V variant on cardiovascular disease have provided inconsistent results. In the LOCAT study, the V162 allele was associated with reduced progression of angiographically assessed diffuse atherosclerosis, whereas the prospective Northwick Park Heart Survey found no impact on the L162V variant on the risk for ischemic heart disease [START_REF] Flavell | Peroxisome proliferator-activated receptor alpha gene variants influence progression of coronary atherosclerosis and risk of coronary artery disease[END_REF]. In a study by Tai et al., 827 men from the Veterans Affair HDL Intervention trial (VA-HIT) showed a reduction of cardiovascular events in V162 carriers only in the presence of insulin resistance or diabetes mellitus [START_REF] Tai | The L162V polymorphism at the peroxisome proliferator activated receptor alpha locus modulates the risk of cardiovascular events associated with insulin resistance and diabetes mellitus: the Veterans Affairs HDL Intervention Trial (VA-HIT)[END_REF]. Some possible limitations should be considered. MI cases from both, the initial and the second sample, were recruited from the Regensburg MI Family Study. Our sampling strategy included MI families from all over Germany with at least two living siblings where the index patient was affected with MI under the age of 60 years and the sibling suffered from MI or severe coronary artery disease. The use of our index criteria could possibly introduce a bias for MI survival and familial form of the disease. However, the fact that all MI cases are from unrelated families of Caucasian ethnicity from all parts of Germany excludes a geographical selection bias. Most important, replication of our results with an independent control group supports the validity of our findings. Yet, we cannot support our findings with prospective data from an unselected sample and hence our results may not be generalizable to the general population.

A second possible limitation is the number of female MI patients included in our study. It is very probable that the lack of statistical power in the female subsample can be attributed to the lower number (~30% of the male subgroup). However, gender-specific effects are unlikely because both, allelic and genotype association tests point to the same direction and have the same order of magnitude, i.e. relative risk reduction of about 20% in male and female MI patients. Furthermore, in none of the previously published studies gender-specific effects could be observed [10;33;34].

We anticipate the possibility that our findings may represent a "false positive" resultthe probably most common problem of many genetic association studies [35]. In particular, our association results did not withstand adjustments for multiple testing.

However, we were able to conduct an independent confirmation study that corroborated our initial findings, which suggest our findings are not a type 1 statistical error. To place our findings in context, there have been two recently conducted whole genome association studies on MI and CAD both employing the Affymetrix GeneChip® Human Mapping 500K Array Set [36]. However, this chip provided no coverage of the region between rs135557, rs135551, and rs135543, where we saw the association signal [36]. In these studies, the remaining PPARα gene region was covered by a total 12 SNP markers, four of which were also included in our work (rs4253662, rs12330015, rs4253754, and rs4253778). Neither the German "Cardiogenics" MI sample nor the British "WTCCC" MI/CAD sample revealed association with any of these SNP markers at a P-value <0.05 [36]. Therefore, the results from these large whole genome screens neither support nor impair our findings.

In conclusion, our results suggest that genetic variations in the PPARα gene, in addition to established genetic and environmental factors, may influence the risk of MI in a European population. Our findings support further investigations to address the role of PPARα in cardiovascular disease and in the future detailed analysis of this gene may be warranted. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

  S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 12 Mar 2008 as manuscript CS20070391 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070391

  genotyping and to analyse gene structure. Control individuals consisted of cardiovascular disease-free married-in spouses, sisters-in-law, and brothers-in-law, from the Regensburg MI Family Study. For the second sample, we identified an additional 667 unrelated MI patients from the Regensburg MI Family Study (age of MI in men <60 years, in women <70 years) and 862 controls from the Third WHO-MONICA Augsburg survey 1994/1995. Individuals with a history of CAD, cerebrovascular accidents (transient ischemic attack or stroke) or peripheral artery disease were excluded from the control group. The MONICA Augsburg survey is a large population-based sample of all German residents of the Augsburg area that consists of individuals aged 25 to 74 years, with about 300 subjects per 10-year age increment and gender. The Augsburg project was part of the international collaborative World Health Organization MONICA study [13]. Written informed consent was obtained from all subjects, and the local ethics committee approved the study. S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 12 Mar 2008 as manuscript CS20070391 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070391

S t a g e 2

 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 12 Mar 2008 as manuscript CS20070391 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070391

  -value of <0.05 indicated statistical significance. Multiple logistic regression analyses were used in the multivariate model of the matched cohort adjusting for age, total cholesterol/HDL cholesterol ratio, systolic blood pressure, body mass index, diabetes mellitus and smoking status with P-values from Effect Wald test. Total cholesterol/HDL cholesterol ratio was chosen because it has been shown to be superior to the use of total cholesterol or LDL cholesterol alone in the prediction of CAD risk and is less affected by ongoing lipid lowering therapy [23-25]. Association results were not corrected for multiple testing. ELECTRONIC DATABASE INFORMATION http://www.ktl.fi/publications/monica/manual/index.htm http://www.hapmap.org/ http://www.ncbi.nlm.nih.gov/SNP/ S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 12 Mar 2008 as manuscript CS20070391 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070391

  in an average marker distance of 5.6 kb. All 17 SNPs had minor allele frequencies (MAF) >0.05 and met Hardy-Weinberg expectations in MI cases and controls in both study samples (P=0.05 level). Haplotypes and their frequencies were constructed from phase-unknown genotype data. A haplotype block was defined as a region in which all pair-wise r² values were >0.4. A total of four haplotype blocks were identified (block A-D, Fig.1). The first haploblock (haploblock A) comprised rs135551, rs135543 and rs135539 and spanned part of intron 2 (6,246 bp); the haploblocks B, C and D were built up by two (rs4253681 and rs7364220), three (rs4823613, rs5766741, rs4253728) and four SNPs (rs4253754, rs4253760, rs4253778, rs11704979) SNPs, respectively. Haploblock B covered part of intron 3, haploblock C spanned most of intron 4, whereas haploblock D commenced within intron 6 and reached across PPARα boundaries into the 3' UTR (Fig.1).

  and P=0.025, OR=0.59, 95% CI=0.38-0.94 for rs135543, respectively; data not shown).

S t a g e 2

 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 12 Mar 2008 as manuscript CS20070391 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070391 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.PPAR-alpha Polymorphisms and Myocardial Infarction 9/21 Reinhard et al.

  S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 12 Mar 2008 as manuscript CS20070391 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070391

  S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 12 Mar 2008 as manuscript CS20070391 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070391Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

  S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 12 Mar 2008 as manuscript CS20070391 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070391Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

  S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 12 Mar 2008 as manuscript CS20070391 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070391Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

S t a g e 2 Figure 1 .

 21 Figure 1. Schematic representation of the PPARα locus. (A) Location of PPARα (NM_005036) and neighbouring genes in a region of ~147 kb on human chromosome 22q13.3. Exon positions are indicated as vertical lines. (B) Relative positions of genotyped SNPs. Position of the 17 SNPs and corresponding rs numbers from dbSNP are shown with arrows. (C) Relative positions of calculated haplotypes. Haploblocks are indicated as block A -D, where block A includes hap1 -hap3 focussed on in the paper. Haplotypes were determined from pair-wise LD comparisons with r² > 0.4 using PHASE version 2.1 [20;21]. (D) Pair-wise r² from HapMap release #20 data. Lighter shading indicates low values (low LD) and darker shading indicates higher values (strong LD). Haploblock A does not reach into the neighbouring gene FLJ27365.

Figure 2 .

 2 Figure 2.Odds ratios for MI depending on the number of hap2 copies in the initial sample (with subjects carrying no hap2 copy as reference group). Individuals carrying 1 or 2 copies of the protective haplotype hap2 are at significantly reduced risk for MI as compared to individuals with no copy of hap2 (overall P-value=0.012).Complete haplotypes could be inferred from 708 individuals.S t a g e 2 ( a ) P O S T -P R I N T

Table 3

 3

	). Hap2 was

P=0.020, OR=0.52, 95% CI=0.23-0.91, respectively, Fig.

2

). Importantly, individuals with 0, 1 or 2 copies of hap2 did not differ with respect to age (54.5 ± 10, 53.8 ± 10.1, 56.8 ± 8.3 years), blood pressure levels (systolic 134 ± 19, 135 ± 17, 136 ± 16 mmHg; diastolic 81 ± 10, 82 ± 9, 82 ± 9 mmHg), body mass index (26.9 ± 3.6, 27.6 ± 4.1, 27.5 ± 4.1 kg/m²), or serum lipid concentrations (LDL cholesterol 146 ± 36 , 148 ± 34, 147 ± 40 mg/dL; HDL cholesterol 54 ± 14, 54 ± 14, 55 ± 15 mg/dL, triglycerides 165 ± 90, 180 ± 112, 180 ± 114 mg/dL respectively). The observed gender difference (71.9, 74.6, 58.1% male, P=0.033) did not influence the results after correction in a logistic regression model (P=0.025, OR=0.48, 95% CI=0.25-0.91).

Table 1 .

 1 TABLES Anthropometric data of 806 individuals from the initial sample and 1,529 individuals from the second sample Clinical Science Immediate Publication. Published on 12 Mar 2008 as manuscript CS20070391 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070391Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

	Initial sample Controls (n=373) 69.2 -59.7 ± 9.4 (32-91) 136 ± 18 82 ± 10 52.4 42.4 239 ± 42 150 ± 33 57 ± 15 182 ± 108 10.7 26.9 ± 3.7 17.4 7.4 59.5 Data are expressed as mean ± standard deviation. BP, blood pressure; LDL, low Second sample MI Cases (n=433) P MI Cases (n=667) Controls (n=862) P Male gender (%) 73.2 n.s. 74.2 70.6 n.s. Age at first MI in years (range) 49.7 ± 7.5 (25-60) -50.3 ± 8.5 (24-69) --Age at inclusion in years (range) 59.4 ± 9.4 (35-82) n.s. 57.6 ± 9.0 (29-82) 50.2 ± 11.9 (25-69) <0.001 Systolic BP (mmHg) 134 ± 18 n.s. 138 ± 19 137 ± 19 n.s. Diastolic BP (mmHg) 81 ± 10 0.043 83 ± 11 83 ± 11 n.s. Hypertension (%) 47.7 n.s. 51.6 43.7 0.003 Anithypertensive therapy (%) 86.6 <0.001 87.1 15.8 <0.001 Total cholesterol (mg/dl) 225 ± 43 <0.001 228 ± 48 237 ± 45 <0.001 LDL cholesterol (mg/dl) 143 ± 36 0.031 156 ± 46 148 ± 43 0.001 HDL cholesterol (mg/dl) 52 ± 14 <0.001 49 ± 13 51 ± 16 0.003 Triglycerides (mg/dl) 162 ± 92 0.006 n.a. n.a. n.a. Lipid lowering therapy (%) 64.2 <0.001 64.8 3.0 <0.001 BMI (kg/m 2 ) 27.5 ± 3.9 0.023 27.3 ± 3.5 27.2 ± 3.9 n.s. Obesity (%) 21.7 n.s. 22.1 20.1 n.s. Type 2 diabetes (%) 16.4 <0.001 15.1 3.5 <0.001 Ever Smoked (%) 74.1 <0.001 72.0 59.3 <0.001 density lipoprotein; HDL, high density lipoprotein; n.a., not available; lipid lowering therapy is defined as regular intake of statins or fibrates, BMI, body mass index; obesity defined as BMI >30 kg/m²; hypertension defined as BP ≥140/90 mmHg; antihypertensive therapy is defined as regular intake of established blood pressure lowering drugs (diuretics, beta-blockers, angiotensin converting enzyme inhibitors, angiotensin 2-blockers, calcium channel blockers, vasodilators, alpha-blockers), type 2 diabetes defined as self-reported diabetes mellitus or regular intake of antidiabetic medication. S t a g e 2 ( a ) P O S T -P R I N T

Table 2 .

 2 Summary of association analyses of the initial case-control sample

Genotype MI cases (n) Genotype controls (n) Allele 2 vs. Allele 1 Armitage's trend test SNP 11 12 22 MAF 11 12 22 MAF OR (95% CI)

									P	OR (95% CI)	P
	rs135557	129 220 80	0.44	100 185 85	0.48	0.86 (0.71-1.05)	0.141	0.86 (0.70-1.05)	0.137
	rs135551	218 183 29	0.28	165 160 45	0.34	0.76 (0.62-0.94)	0.013	0.74 (0.61-0.94)	0.012
	rs135543	197 194 40	0.32	158 158 54	0.36	0.83 (0.68-1.02)	0.079	0.81 (0.68-1.02)	0.082
	rs135539	137 208 83	0.44	102 194 75	0.46	0.90 (0.74-1.09)	0.285	0.90 (0.73-1.09)	0.281
	rs881740	330 93	7	0.12	299 69	3	0.10	1.26 (0.92-1.73)	0.142	1.30 (0.92-1.70)	0.141
	rs4253662	357 63	4	0.08	289 56	8	0.10	0.81 (0.57-1.14)	0.215	0.79 (0.59-1.14)	0.234
	rs4253681	301 121 3	0.15	279 86	3	0.13	1.23 (0.92-1.64)	0.160	1.23 (0.93-1.70)	0.139
	rs7364220	260 148 18	0.22	246 114 11	0.18	1.23 (0.96-1.57)	0.104	1.24 (0.96-1.59)	0.100
	rs12330015 337 79	2	0.10	302 62	3	0.09	1.08 (0.77-1.51)	0.656	1.06 (0.77-1.53)	0.651
	rs4823613	208 181 35	0.30	196 147 24	0.27	1.16 (0.93-1.50)	0.181	1.17 (0.93-1.46)	0.176
	rs5766741	217 170 36	0.29	196 152 21	0.26	1.12 (0.90-1.40)	0.303	1.17 (0.90-1.41)	0.298
	rs4253728	217 179 34	0.29	198 151 21	0.26	1.14 (0.92-1.42)	0.238	1.17 (0.92-1.44)	0.230
	rs1800206	380 45	2	0.06	328 42	1	0.06	0.97 (0.64-1.47)	0.870	0.99 (0.64-1.47)	0.871
	rs4253754	265 147 15	0.21	239 118 12	0.19	1.10 (0.86-1.40)	0.460	1.09 (0.86-1.42)	0.452
	rs4253760	274 133 17	0.20	240 114 14	0.19	1.03 (0.80-1.32)	0.841	1.03 (0.80-1.32)	0.842
	rs4253778	272 137 15	0.20	238 113 15	0.20	1.01 (0.79-1.30)	0.937	0.99 (0.79-1.30)	0.937
	rs11704979 324 96	6	0.13	294 71	5	0.11	1.18 (0.87-1.61)	0.287	1.16 (0.87-1.61)	0.287
	For the PPARα locus, 17 genotyped SNPs and their rs numbers from dbSNP
	database are shown. Numbers (n) for each genotype (11/12/22) and minor allele
	frequencies (MAF) are listed separately for MI cases and controls. Odds ratios (OR)
	with 95% confidence interval (CI) and P-values are given for allelic association and
	genotypic association based on the Armitage test for trend with significant P-values
	in bold print.

S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 12 Mar 2008 as manuscript CS20070391 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070391

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Table 3 .

 3 Frequencies of hap1 -hap3, and association results with MI in the initial and second case-control samples adj P-value from multivariate model (adjusted for age, total cholesterol/HDL cholesterol levels, systolic blood pressure, body mass index, diabetes mellitus, smoking). *From the initial, second and matched sample n=98, n=41, and n=113 individuals were excluded due to improbable haplotype reconstruction. Significant Pvalues are presented in bold print.

	Haploblock A	hap1	hap2	hap3
	Initial sample (n=708)*
	No of haplotypes (frequency) MI Cases	424 (0.55)	197 (0.26)	104 (0.14)
	Controls	345 (0.53)	210 (0.32)	70 (0.11)
	OR (95% CI) 1.11 (0.78-1.59) 0.76 (0.56-1.02) 1.29 (0.90-1.84)
	P	0.549	0.067	0.167
	Second sample (n=1488)*
	No of haplotypes (frequency) MI Cases	727 (0.56)	312 (0.24)	198 (0.15)
	Controls	884 (0.53)	460 (0.27)	238 (0.14)
	OR (95% CI) 1.20 (0.93-1.55) 0.80 (0.65-0.98) 1.06 (0.84-1.33)
	P	0.159	0.034	0.643

P

Table 4 .

 4 Summary of association analyses of the second case-control sample

Genotype MI cases (n) Genotype controls (n) Allele 2 vs. Allele 1 Armitage's trend test SNP 11 12 22 MAF 11 12 22 MAF OR (95% CI) P OR (95% CI) P

  For the PPARα locus, 4 genotyped SNPs and their rs numbers from dbSNP database are shown. Numbers (n) for each genotype (11/12/22) and minor allele frequencies (MAF) are listed separately for MI cases and controls. Odds ratios (OR) with 95% confidence interval (CI) and P-values are given for allelic association and genotypic association based on the Armitage test for trend with significant P-values in bold print.

	rs135557	192 345 121 0.45	247 432 176 0.46	0.95 (0.82-1.10)	0.496	0.95 (0.82-1.10)	0.488
	rs135551	365 253 49	0.26	407 359 67	0.30	0.85 (0.72-0.99)	0.047	0.87 (0.72-0.99)	0.046
	rs135543	342 260 61	0.29	383 384 91	0.33	0.82 (0.70-0.96)	0.014	0.84 (0.71-0.96)	0.014
	rs135539	214 334 116 0.43	246 430 175 0.46	0.88 (0.76-1.00)	0.078	0.87 (0.76-1.01)	0.075

ABBREVIATIONS

PPARα -Peroxisome proliferator activated receptor alpha SNP -single nucleotide polymorphism MI -myocardial infarction LD -linkage disequilibrium CAD -coronary artery disease OR -odds ratio MAF -minor allele frequency S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 12 Mar 2008 as manuscript CS20070391 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070391 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 12 Mar 2008 as manuscript CS20070391 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070391

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ACKNOWLEDGEMENT

This study was supported by grants from the Deutsche Forschungsgemeinschaft (He1921/9-1), the Vaillant-Stiftung (C.H., H.S.), and the "Deutsche Stiftung fuer Herzforschung." The KORA research platform (KORA: Cooperative Research in the Region of Augsburg) and the MONICA Augsburg studies (Monitoring trends and determinants on cardiovascular diseases) were initiated and financed by the GSF -National Research Centre for Environment and Health. The MONICA Augsburg Study was initiated and conducted by Ulrich Keil and co-workers. KORA-gen was supported within the German National Genomic Research Network (NGFN) and by the Federal Ministry of Education and Research (BMBF). S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 12 Mar 2008 as manuscript CS20070391 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070391 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

PPAR-alpha Polymorphisms and Myocardial Infarction 14/21 Reinhard et al.

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