Quinapril Treatment Increases Insulin-Stimulated Endothelial Function ...

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The Journal of Clinical Endocrinology & Metabolism 91(3):1001–1008 Copyright © 2006 by The Endocrine Society doi: 10.1210/jc.2005-1231

Quinapril Treatment Increases Insulin-Stimulated Endothelial Function and Adiponectin Gene Expression in Patients with Type 2 Diabetes Thomas S. Hermann, Weijie Li, Helena Dominguez, Nikolaj Ihlemann, Christian Rask-Madsen, Atheline Major-Pedersen, Dorthe Baunbjerg Nielsen, Kaj Winther Hansen, Meredith Hawkins, Lars Kober, and Christian Torp-Pedersen Department of Cardiology Y, Bispebjerg Hospital (T.S.H., H.D., N.I., A.M.-P., D.B.N., C.T.-P.), 2400 Copenhagen, Denmark; Division of Endocrinology and Diabetes Research and Training Center, Albert Einstein College of Medicine (W.L., M.H.), Bronx, New York 10461; Section on Vascular Cell Biology and Complications, Joslin Diabetes Center (C.R.-M.), Boston, Massachusetts 02215; Department of Clinical Biochemistry, Gentofte University Hospital (K.W.H.), DK-2900 Gentofte, Denmark; and Department of Cardiology B, Rigshospitalet Heart Center (L.K.), DK-2100 Copenhagen, Denmark Objective: Angiotensin-converting enzyme inhibitors reduce cardiovascular mortality and improve endothelial function in type 2 diabetic patients. We hypothesized that 2 months of quinapril treatment would improve insulin-stimulated endothelial function and glucose uptake in type 2 diabetic subjects and simultaneously increase the expression of genes that are pertinent for endothelial function and metabolism. Methods: Twenty-four type 2 diabetic subjects were randomized to receive 2 months of quinapril 20 mg daily or no treatment in an open parallel study. Endothelium-dependent and -independent vasodilation was studied during serotonin or sodium nitroprusside infusion in the diabetic patients and in 15 healthy subjects. Endothelial function, insulin-stimulated endothelial function, and insulin-stimulated glucose uptake were measured before and after quinapril treatment.

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NHIBITORS OF ANGIOTENSIN-converting enzyme (ACE) reduce the incidence of cardiovascular disease in patients with type 2 diabetes (1). Endothelial dysfunction, an early marker of cardiovascular disease, is present in patients with insulin resistance and type 2 diabetes, and these patients also have impaired insulin-stimulated endothelial function, compared with lean healthy subjects (2, 3). Consequently, reversal of endothelial dysfunction may be important for the prevention of cardiovascular events. ACE inhibition improves endothelial function in patients with types 1 and 2 diabetes by a nitric oxide-dependent mechanism (4, 5). In vivo studies have demonstrated that ACE inhibition decreases the concentration of the vasoconstrictor endothelin-1 (6) and increases the gene expression of endothelial nitric oxide synthase in vascular tissue (7). ACE inhibition reduces the degree of atherosclerosis in a diabetic rat model with accelerated atherosclerosis (8) and inhibits atherosclerosis in type 2 diabetic patients, measured by reduced carotid intima-media thickness (9).

First Published Online December 13, 2005 Abbreviations: ACE, Angiotensin-converting enzyme; CRP, C-reactive protein; IRS, insulin receptor substrate; l-NMMA, NG-monomethyll-arginine; NO, nitric oxide; PI 3, phosphatidylinositol 3. JCEM is published monthly by The Endocrine Society (http://www. endo-society.org), the foremost professional society serving the endocrine community.

Blood flow was measured by venous occlusion plethysmography. Gene expression was measured by real-time PCR. Results: Quinapril treatment increased insulin-stimulated endothelial function in the type 2 diabetic subjects (P ⫽ 0.005), whereas forearm glucose uptake was unchanged. Endothelial function was also increased by quinapril (P ⫽ 0.001). Systolic and diastolic blood pressures were reduced by quinapril (P ⬍ 0.001). Quinapril increased adiponectin gene expression in vascular tissue obtained from sc adipose biopsies. Conclusions: Quinapril treatment increases insulin-stimulated endothelial function in patients with type 2 diabetes. Increased vascular adiponectin gene expression may contribute to this beneficial effect. (J Clin Endocrinol Metab 91: 1001–1008, 2006)

ACE inhibition is associated with a lower rate of new-onset type 2 diabetes in high-risk patients with cardiovascular disease (10, 11). Some physiological studies support the concept that ACE inhibition might prevent type 2 diabetes by improving whole-body insulin sensitivity (12–14), whereas others have shown that ACE inhibition has no effect on insulin sensitivity (15, 16). Angiotensin II is suggested to induce insulin resistance (17), whereas antagonism of angiotensin II ameliorates insulin resistance (18). The beneficial effects of ACE inhibition on both endothelial function and insulin resistance could be explained by increased plasma levels of adiponectin (19). Indeed, this adipose-derived circulating protein improves insulin action in conscious mice, and its levels are tightly correlated with insulin sensitivity in animals and humans (20). Hypoadiponectinemia is associated with endothelial dysfunction (21), and adiponectin increases nitric oxide production in endothelial cells (22, 23). Given these observations, we hypothesized that treatment with quinapril would increase insulin stimulated forearm glucose uptake and insulin-stimulated endothelial function. Furthermore, because ACE inhibitors increase adiponectin levels and the latter have favorable effects on insulin action, we examined the impact of quinapril on adiponectin gene expression in patients with type 2 diabetes.

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J Clin Endocrinol Metab, March 2006, 91(3):1001–1008

Patients and Methods Twenty-four patients with type 2 diabetes and no previous history of cardiovascular disease, retinopathy, or nephropathy were included. All participants underwent a screening, which included measurement of blood pressure, body weight, fasting glucose, fasting cholesterol, and a 2-h oral glucose challenge (75 g glucose). Patients were randomized (computer-generated random numbers in sealed envelopes) to receive 2 months tablet treatment with 20 mg quinapril daily (Accupro, Pfizer, Copenhagen, Denmark) or no treatment in an open parallel group study. The allocation ratio (of patients receiving quinapril to patients receiving no treatment) was 2:1. Measurement of endothelium-dependent vasodilation, endothelium-independent vasodilation, insulin-stimulated endothelium-dependent vasodilation, insulin-stimulated forearm glucose uptake, and an oral glucose challenge (on a separate day) was performed before and after 2 months in each group. One type 2 diabetic subject in the treatment group was excluded due to noncompliance and a change in smoking status from nonsmoker to heavy smoking shortly after the first examination. During quinapril treatment one subject developed a rash and three subjects developed a dry cough. Fifteen healthy subjects matched with regard to age and smoking status were used as a healthy control group. The three groups are designated in the following groups: 1) type 2 diabetic treatment group (type 2 diabetic patients randomized to quinapril treatment); 2) type 2 diabetic control group (type 2 diabetic patients randomized to no treatment); and 3) healthy control group (healthy control population). The study was approved by the local ethical committee and the Danish Medicines Agency. Participants gave their written informed consent. All measurements in this study were performed blinded to the treatment protocol. All studies were started at 0800 h after an 8-h overnight fast, which included abstinence from smoking. Experimental procedures and measurement of blood flow was performed as previously described (3). Blood flow was measured by venous occlusion plethysmography. Assessment of forearm vascular function was performed by separately stimulation of endothelium-dependent vasodilation and endotheliumindependent vasodilation. Both measures are necessary for the measurement of vascular function and are also suitable for the evaluation of action and effect of cardiovascular drugs. In the current study, endothelial nitric oxide (NO) production was stimulated by infusion of serotonin. Serotonin is an agonist of endothelial NO production and is highly NO specific. Endothelium-independent vasodilation (vascular smooth muscle relaxation) was tested by infusion of sodium nitroprusside, which acts as an external NO donor in vascular smooth muscle cells. Local insulin infusion in the brachial artery for 60 min was used. Blood flow was expressed as (milliliters per 100 ml forearm per minute). Insulin stimulation of endothelium-dependent vasodilation was calculated as percent increase in blood flow from the serotonin response. For the study of gene expression of ACE, adiponectin, insulin receptor substrate (IRS)-1, phosphatidylinositol 3 (PI 3)-kinase, and Akt-1 during quinapril treatment, vascular tissue was isolated from sc fat biopsies, obtained from the study subjects before and after the 2 months. The isolation of vascular tissue from adipose tissue was performed as described by Jiang (24). After isolation, the vascular tissue was immediately frozen in liquid nitrogen and stored at ⫺80 C. From the biopsy samples obtained as described above, total RNA was extracted and gene expression was measured by real-time PCR as previously described (25). Primers used were as follows: glyceraldehyde3-phosphate dehydrogenase forward, TCGGAGTCAACGGATTTG, reverse, GCATCGCCCCACTTGATT; adiponectin forward, GGTGGGCTCCTTACAGAACA, reverse, TTCAAAGCATCACAGGACCA; IRS-1

FIG. 1. Infusion protocol. The infusion protocol shows the infusion schedule of serotonin (5-HT), sodium nitroprusside (SNP), L-NMMA, and insulin.

Hermann et al. • Quinapril and Insulin-Stimulated Endothelium

forward, AGTCCCAGCACCAACAGAAC, reverse, TCATCCGAGGAGATGAAACC; Akt-1 forward, CCCTTCTACAACCAGGACCA, reverse, ACACGATACCGGCAAAGAAG; PI 3-kinase forward, TCATATTGACTTCGGGCACA, reverse, TCAGCATCATGGAGAACAGG; and ACE forward, TTGACAAGATCGCCTTTATCC, reverse, GTAAGGCACGCTAGAAGGAAT. Results are expressed as fold change in gene expression by determining the ratio of copy number of the gene of interest in a given individual after quinapril vs. placebo treatment, corrected for expression of glyceraldehyde-3-phosphate dehydrogenase in the samples. Vascular tissue was obtained from six subjects in the type 2 diabetic treatment group and two subjects in the type 2 diabetic control group. The purity of the isolated vascular stroma was assessed by light microscopy after staining with hematoxylin and eosin and also immunohistochemical staining with CD34.

Infusion protocol In the study of endothelium-dependent vasodilation, incremental doses of serotonin (7, 21, and 70 ng/min; Clinalfa, La¨ufelfingen, Switzerland) were infused (Fig.1). Each dose was infused for 5 min to obtain a stable blood flow. After at least 30 min washout of serotonin, when blood flow had returned to baseline, the effect of insulin on endothelium-independent vasodilation was assessed by infusion of the NO donor sodium nitroprusside (Nitropress, Abbott Laboratories, North Chicago, IL) in doses of 0.5, 1, and 1.5 ␮g/min. The doses were chosen from previous experience in attempt to match flow levels induced by serotonin. The sodium nitroprusside studies were performed in all study subjects. Insulin-stimulated endothelial function was assessed by intraarterial insulin infusion [Actrapid (Novo Nordisk Scandinavia, Malmo¨, Sweden) in 1% human albumin solution (vehicle)] at a rate of 0.05 mU ⫻ kg body weight⫺1 ⫻ min⫺1. Insulin was continuously infused for 60 min, and a dose response study of serotonin was subsequently performed immediately afterward. The fraction of NO-mediated vasodilation during serotonin-insulin infusion at 60 min forearm hyperinsulinemia was determined in 11 subjects in the type 2 diabetic treatment group and five subjects in the type 2 diabetic control group after 10 min intraarterial infusion of NGmonomethyl-l-arginine (l-NMMA; Clinalfa) in a dose of 3.3 mg/min, followed by a serotonin dose-response study.

Glucose uptake and measurement of insulin concentration Blood samples were drawn simultaneously from the artery and vein in the infused arm. The plasma concentration of glucose was determined by the glucose oxidase method (Vitros; Johnson & Johnson, New Brunswick, NJ), and serum insulin concentration was determined by a microparticle enzyme immunoassay (Axsym Insulin B2D010; Abbott). Insulin-stimulated glucose uptake in the forearm was calculated as previously described (26). Glucose uptake was assessed every 10 min during insulin infusion. During the oral glucose challenge, blood samples for measurement of plasma glucose and serum insulin were collected at time 0, 15, 30, 60, and 120 after digestion of 75 g glucose. The concentration of adiponectin was determined by RIA (human adiponectin RIA kit, Linco Research, St. Charles, MO).

Statistical analyses Comparison of blood flow values were performed with mixed models using the PROC MIXED procedure in the Statistical Analysis Software (version 8.2; SAS Institute, Cary, NC). For studies of blood flow, loga-



rithmic transformation of flow was used to satisfy the model assumption (homogeneity of variance). The dose-response studies entered the model as fixed effects as did the interaction between dose-response study and dose of vasodilator. Study subject and the interaction between study subject and dose of vasodilator entered the model as random effects. The level of statistical significance was chosen as P ⱕ 0.05 (two-sided test). Continuous variables were normally distributed and are presented as means ⫾ sd (in figures as mean and sem).

Results Characteristics of the study population

Characteristics are shown in Table 1. Type 2 diabetic subjects had significant higher fasting plasma glucose, fasting serum insulin, C-reactive protein (CRP) concentration, and systolic blood pressure than the healthy controls. No significant change in the CRP or adiponectin concentration was seen in the type 2 diabetic treatment group or the type 2 diabetic control group during the study. Systolic and diastolic blood pressure and heart rate

Systolic and diastolic blood pressure was reduced significantly (systolic blood pressure: P ⬍ 0.0001 after vs. before; diastolic blood pressure P ⬍ 0.0001 after vs. before) in the type 2 diabetic treatment group (Fig. 2). In the type 2 diabetic control subjects, systolic blood pressure was not altered, whereas a significant reduction in diastolic blood pressure was observed (P ⬍ 0.0001; Fig. 2). Dose-response studies of serotonin and sodium nitroprusside did not cause significant changes in systolic blood pressure or diastolic blood pressure. Heart rate was not changed in the type 2 diabetic treatment group or the type 2 diabetic control group (Table

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1), and the heart rate was also unchanged during doseresponse studies of serotonin and sodium nitroprusside and during insulin infusion. Oral glucose challenge

Fasting plasma glucose was not altered in the type 2 diabetic treatment group (before ⫽ 10.8 ⫾ 2.7 mm vs. after ⫽ 9.8 ⫾ 1.9 mm, NS). Fasting serum insulin concentration did not change (before ⫽ 18.5 ⫾ 12.8 mU/liter vs. after ⫽ 16.7 ⫾ 9.3 mU/liter, NS). In the type 2 diabetic control group, no significant change in fasting plasma glucose (before ⫽ 9.5 ⫾ 3.4 mm vs. after ⫽ 8.3 ⫾ 1.7 mm, NS) or fasting serum insulin (before ⫽ 15.0 ⫾ 9.6 mU/liter vs. after ⫽ 14.5 ⫾ 10.2 mU/liter, NS) was seen. Quinapril treatment changed neither the plasma glucose concentration (before ⫽ 17.2 ⫾ 2.7 mm vs. after ⫽ 17.2 ⫾ 2.2 mm, NS) nor the serum insulin concentration (before ⫽ 41.8 ⫾ 25.7 mU/liter vs. after ⫽ 42.4 ⫾ 23.8 mU/liter, NS) at 2 h after the challenge. Quinapril treatment did not cause significant changes in plasma glucose or serum insulin concentration 15, 30, or 60 min after the challenge. In the type 2 diabetic control group, no significant changes were seen in the postchallenge values of glucose or insulin during the 2 months. Endothelium-dependent and -independent vasodilation

Subjects with type 2 diabetes had a significantly lower serotonin response than healthy controls (Fig. 3A). Baseline blood flow was not increased by quinapril treatment (be-

TABLE 1. Characteristics of the quinapril-treated group, the time control group, and the healthy controls T2DM treatment group

N Age (yr) Sex (M/F) Smoking (%) Oral hypoglycemic drugs (%) Metformin Glibenclamid/glimepirid Metformin ⫹ glibenclamid Aspirin (%) Diuretics (%) BMI (kg/m2) Systolic BP (mm Hg) Diastolic BP (mm Hg) Heart rate (beats/min) Plasma glucose (mM) Serum insulin (mU/liter) HbA1c (%) Total cholesterol (mM) LDL cholesterol (mM) HDL cholesterol (mM) Triglycerides (mM) Potassium (mM) CRP (g/liter) Adiponectin (␮g/liter)

T2DM control group

Before quinapril

After quinapril for 2 months

Before no treatment

After no treatment for 2 months

15 57.5 ⫾ 1.4 9/6 7 (47) 13 (87) 6 6 1 2 (13) 4 (27) 30.3 ⫾ 5.7 136 ⫾ 16 64 ⫾ 8 66 ⫾ 12 10.8 ⫾ 2.7 18.5 ⫾ 12.8 7.6 ⫾ 0.8 4.9 ⫾ 1.2 3.0 ⫾ 0.8 1.1 ⫾ 0.4 2.1 ⫾ 1.9 4.0 ⫾ 0.4 7.3 ⫾ 6.2 5.9 ⫾ 3.1

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8 54.3 ⫾ 2.9 5/3 4 (50) 4 (50) 3 1

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1 (13) 0 30.8 ⫾ 3.8 146 ⫾ 15 70 ⫾ 11 69 ⫾ 11 9.5 ⫾ 3.4 15.0 ⫾ 9.6 7.7 ⫾ 1.7 4.8 ⫾ 0.8 2.8 ⫾ 0.8 1.1 ⫾ 0.3 2.8 ⫾ 3.1 4.2 ⫾ 0.3 3.8 ⫾ 2.5 5.3 ⫾ 4.5

29.4 ⫾ 3.6 147 ⫾ 23 67 ⫾ 11 68 ⫾ 14 8.3 ⫾ 1.7 14.5 ⫾ 10.2 7.2 ⫾ 1.4 4.7 ⫾ 1.1 2.8 ⫾ 1.1 1.2 ⫾ 0.6 2.1 ⫾ 1.4 4.1 ⫾ 0.6 4.6 ⫾ 2.2 5.4 ⫾ 3.4

6 6 1 30.2 ⫾ 6.5 129 ⫾ 16b 59 ⫾ 12b 69 ⫾ 15 9.8 ⫾ 1.9 16.7 ⫾ 9.3 7.6 ⫾ 0.8 4.8 ⫾ 1.2 2.9 ⫾ 1.2 1.1 ⫾ 0.4 2.4 ⫾ 3.0 4.1 ⫾ 0.4 6.5 ⫾ 6.2 5.5 ⫾ 2.3

Healthy control group

15 53.7 ⫾ 2.6 10/5 5 (33) 0 0 0 0 0 0 24.0 ⫾ 2.9a 121 ⫾ 16a 62 ⫾ 12 62 ⫾ 12 5.2 ⫾ 0.8a 4.8 ⫾ 1.1a 5.3 ⫾ 0.8a 4.8 ⫾ 1.2 2.9 ⫾ 1.2 1.4 ⫾ 0.4a 1.2 ⫾ 0.8a 3.9 ⫾ 0.4 1.7 ⫾ 2.3a

Data are presented as mean ⫾ SD. M, Male; F, female; BP, blood pressure; BMI, body mass index; T2DM, type 2 diabetes mellitus; HbA1c, glycosylated hemoglobin; LDL, low-density lipoprotein; HDL, high-density lipoprotein. a P ⬍ 0.01 healthy controls vs. type 2 diabetic study subjects. b P ⬍ 0.05 after quinapril vs. before quinapril treatment.

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FIG. 2. Systolic and diastolic blood pressure in the type 2 diabetic treatment group and the type 2 diabetic control group. Systolic and diastolic blood pressure was significantly reduced in the treatment group. Systolic blood pressure after treatment (white circles) was reduced by 7 mm Hg, compared with systolic blood pressure before treatment (black circles) (P ⬍ 0.001), and diastolic blood pressure after treatment (white triangles) was reduced by 4 mm Hg, compared with diastolic blood pressure before quinapril (black triangles) (P ⬍ 0.001). In the type 2 diabetic control group, no significant change in systolic blood pressure was seen (black squares before and white squares after), whereas a change of 3.5 mm Hg in diastolic blood pressure was seen (P ⫽ 0.001). No change in blood pressure was seen during dose-response studies of serotonin (5-HT) and sodium nitroprusside (SNP). T2DM, Type 2 diabetes; INS, insulin.

fore ⫽ 1.75 ⫾ 0.40 vs. after ⫽ 1.89 ⫾ 0.71 ml per 100 ml per minute, P ⫽ 0.4). The serotonin response was increased significantly, by an average of 16.5% at all three dose levels, P ⫽ 0.001 vs. pretreatment (Fig. 3A). Endothelium-independent vasodilation, assessed during sodium nitroprusside infusion (Fig. 3B), was lower in the type 2 diabetic subjects than the healthy controls but the sodium nitroprusside response was unchanged by quinapril treatment (P ⫽ 0.8 before vs. after treatment). The sodium nitroprusside response was also unchanged in the type 2 diabetic control group. At the beginning the sodium nitroprusside response was 2.31 ⫾ 0.72 (dose 1), 5.00 ⫾ 1.73 (dose 2), and 7.41 ⫾ 3.04 (dose 3), and after the 2 months, the response was 2.03 ⫾ 0.71 (dose 1), 5.60 ⫾ 3.00 (dose 2), and 7.95 ⫾ 2.06 (dose 3). Insulin-stimulated endothelial function

The insulin-stimulated serotonin response was markedly reduced in the subjects with type 2 diabetes, compared with the healthy control group (Fig. 4A). Quinapril treatment increased the insulin-stimulated serotonin response significantly (Fig. 4A; P ⬍ 0.001 vs. pretreatment). To correct for the increase in endothelium-dependent vasodilation during treatment, we calculated the percentage increase in serotonin response by insulin. After this correction, insulin-stimulated endothelial function was still significantly increased after 2 months of quinapril treatment (Fig. 4B; P ⫽ 0.005 after treatment vs. before treatment). The degree of NO-mediated vasodilation during insulin-serotonin infusion was assessed during coinfusion of l-NMMA. The blood flow increase induced by serotonin-insulin coinfusion was abolished by lNMMA infusion. Before quinapril, blood flow during the serotonin-insulin-l-NMMA coinfusion was 1.55 ⫾ 0.42 (dose 1), 1.83 ⫾ 0.52 (dose 2), and 1.83 ⫾ 0.65 (dose 3), which was not altered after quinapril treatment: 1.85 ⫾ 0.25 (dose 1),

1.86 ⫾ 0.16 (dose 2), and 1.93 ⫾ 0.26 (dose 3) (P ⫽ 0.4 after treatment vs. before treatment). In the type 2 diabetic control group, the serotonin response was slightly but not significantly increased (P ⫽ 0.2), and the insulin-stimulated serotonin response was unchanged after 2 months. Insulin-stimulated forearm glucose uptake

Forearm glucose uptake was assessed during 60 min of intraarterial insulin infusion. Local serum insulin was increased from 9.8 ⫾ 6.0 to 251.2 ⫾ 86.2 mU/liter after 10 min of insulin infusion (P ⬍ 0.0001). Local serum insulin concentration at 30 min infusion was 252.1 ⫾ 77.7, and after 60 min insulin infusion concentration was 259.7 ⫾ 85.8 mU/ liter. During the 60 min intraarterial insulin infusion, no change in systemic insulin was seen. In the type 2 diabetic treatment group, systemic serum insulin was 11.2 ⫾ 7.2 mU/ liter before insulin infusion and was 12.6 ⫾ 8.3 mU/liter after 60 min insulin (P ⫽ 0.6) Baseline and insulin-stimulated forearm glucose uptake was not altered by quinapril treatment (Fig. 5). Effect of quinapril on adiponectin, IRS-1, PI 3-kinase, Akt1, and ACE gene expression in vascular and adipose tissue

Quinapril treatment increased the gene expression ratio of adiponectin in the vascular tissue by 2-fold, from 0.150 ⫾ 0.058 before to 0.350 ⫾ 0.205 after treatment (P ⫽ 0.048 before vs. after). In the control group, samples from only two subjects were available. In this group adiponectin expression was 0.153 ⫾ 0.118 before and 0.191 ⫾ 0.159 after 2 months. Gene expression of IRS-1 (before ⫽ 0.060 ⫾ 0.054 vs. after quinapril ⫽ 0.030 ⫾ 0.004, NS), Akt-1 (before ⫽ 0.069 ⫾ 0.044 vs. after quinapril ⫽ 0.043 ⫾ 0.035, NS), and PI 3-kinase


FIG. 3. A, Serotonin response in 15 patients with type 2 diabetes before (black squares) and after 2 months of quinapril treatment (black triangles) and in 15 healthy control subjects (black circles). The serotonin response in the patients with type 2 diabetes was significantly increased after 2 months of quinapril treatment (P ⫽ 0.001). The serotonin response in the subjects with type 2 diabetes was significantly lower than in the healthy subjects (P ⬍ 0.001). The L-NMMA response in the quinapril group before treatment (white squares) was not changed after quinapril treatment (white triangles) (P ⫽ 0.5). B, Sodium nitroprusside response in the healthy controls (black squares) and the type 2 diabetic subjects before (black circles) and after (white circles) 2 months of quinapril treatment. No significant change in the sodium nitroprusside response was seen (P ⫽ 0.8). T2DM, Type 2 diabetes.

(before ⫽ 0.0011 ⫾ 0.0007 vs. after quinapril ⫽ 0.0010 ⫾ 0.0008, NS) was unchanged by quinapril. The expression of the ACE gene was not significantly changed (before ⫽ 0.031 ⫾ 0.027 vs. after quinapril ⫽ 0.021 ⫾ 0.008, NS). In adipose tissue, relative gene expression of adiponectin was 25-fold higher than the expression ratio seen in vascular tissue. Adiponectin gene expression was unchanged by quinapril in adipose tissue, from 3.69 ⫾ 0.57 before quinapril to 3.45 ⫾ 0.68 after quinapril (NS).


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FIG. 4. A, Insulin enhanced serotonin response in healthy subjects (white circles) and type 2 diabetic subjects (T2DM) before (white squares) and after (upward-slanting white triangles) 2 months of quinapril treatment and in the control group before (white diamonds) and after 2 months (downward-slanting white triangles). The serotonin response in the healthy subjects (black circles) and type 2 diabetic subjects before quinapril (black squares) and after 2 months of quinapril treatment (black squares) and in the type 2 diabetic control group before (black diamonds) and after 2 months (downward-slanting black triangles) is shown for comparison. Insulin-enhanced serotonin response was significantly increased by quinapril (P ⬍ 0.001), and the type 2 diabetic subjects had a lower insulin enhancement of the serotonin response than the healthy controls. In the type 2 diabetic control group, the serotonin response was increased to some extent after the 2 months, but the change was not significant (P ⫽ 0.2). The insulin-enhanced serotonin response in the type 2 diabetic control group was not significantly increased during the 2 months. The percent enhancement of the serotonin response by insulin is shown (B). After quinapril treatment the enhancement of the serotonin response by insulin was significantly increased (white squares), compared with before quinapril treatment (white triangles) (P ⫽ 0.005).

Discussion

We report that 2 months of quinapril treatment improved insulin-stimulated endothelial function without changing insulin-stimulated glucose uptake in the forearm or postchallenge glucose or insulin levels in patients with type 2

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FIG. 5. Forearm uptake during 60 min of insulin infusion. No significant change in glucose uptake was seen in the type 2 diabetic (T2DM) treatment group or the type 2 diabetic time control group.

diabetes. Furthermore, adiponectin gene expression was increased in vascular tissue from the quinapril-treated patients. To our knowledge, we are the first to demonstrate a positive effect of ACE inhibition on insulin-stimulated endothelial function in patients with type 2 diabetes, and the observed increase in adiponectin gene expression could be one possible underlying mechanism. The observations in the current study are not regarded as being specific to quinapril but are also expected to be reproduced with other nonsulfhydrylcontaining ACE inhibitors. The vascular system in patients with insulin resistance and type 2 diabetes is characterized by endothelial dysfunction (2). The improvement in endothelial function with ACE inhibition in our study is consistent with observations in diverse patient populations, including type 1 or type 2 diabetes, coronary heart disease, or heart failure (4, 5, 27, 28). Type 2 diabetic patients have increased plasma concentration and also increased endogenous activity of the potent vasocon-


strictor endothelin-1 (29, 30). There is existing evidence that endothelin-1 is reduced by ACE inhibition (6, 29), which could be one explanation for the observed increase in endothelial function. Also, ACE inhibition reduces the concentration of the endogenous NO synthase inhibitor asymmetric dimethylarginine in type 2 diabetic patients (31), which could facilitate NO-dependent vasodilation. In our study we did not observe any change in endothelium-independent vasodilation, suggesting that the smooth muscle cell sensitivity to NO was not increased. We studied the effect of quinapril in subjects with type 2 diabetes with a high incidence of smoking. Patients with type 2 diabetes have increased oxidant stress, which is also present in smokers. Because ACE inhibitors are known to reduce oxidant stress (32), this could also mediate some beneficial effect on NO-mediated endothelial function in the current study. However, we did not measure markers of oxidative stress. In this study the insulin-stimulated serotonin response was significant increased and was also significant increased after adjustment for the enhancement of the serotonin response by quinapril. Furthermore, blood flow during insulin-serotonin coinfusion was inhibited by l-NMMA before and after quinapril treatment, which indicates that insulinstimulated vasodilation was mediated by NO and that the observed increase in insulin-stimulated vasodilation during quinapril treatment was also mediated by NO. It is well acknowledged that insulin causes enhancement of endothelial-dependent vasodilation in resistance vessels (33) and increases capillary recruitment in healthy subjects (34). In resistance vessels, insulin stimulates vasodilation by stimulating IRS-1-associated PI 3-kinase activity, which is followed by activation of Akt and an increase in the activity of endothelial nitric oxide synthase (35). This pathway is blunted in vascular tissue in an animal model of insulin resistance (24). In type 2 diabetic subjects receiving hypoglycemic drug therapy, improvement of whole-body insulin sensitivity and insulin-stimulated vasodilation occurs within the same time range (3, 36), and therefore, a relation is suggested to exist. In our study no change in forearm glucose uptake or whole-body glucose uptake was seen, whereas insulin-stimulated vasodilation was significantly increased. This indicates that a change in insulin-stimulated vasodilation is not necessarily preceded or accompanied by an increase in whole-body insulin sensitivity. Of note, our studies did not include a euglycemic hyperinsulinemic clamp, and therefore, it is possible that we may not have been able to detect subtle changes in whole-body glucose uptake. However, because both the forearm glucose uptake and the glucose and insulin responses to the oral glucose challenge remained unchanged during quinapril treatment, further intensive studies of glucose metabolism do not appear to be warranted. The lack of an effect on glucose metabolism by quinapril could be due to the dosage or an insufficient duration of treatment. In a study with captopril, the effect on glucose metabolism was seen already after 2 d of treatment (12). However, the dosage used in the current study may have permitted the detection of a discrepancy among responses by tissue type. Adiponectin gene expression was increased in vascular


biopsies from the quinapril-treated subjects, whereas no change was seen in gene expression of IRS-1, PI 3-kinase, or Akt-1. Adiponectin is abundantly expressed in adipose tissue in humans and is present in a high concentration in plasma. Subjects with obesity and type 2 diabetes have a lower plasma level of adiponectin than healthy subjects (37). A low concentration of adiponectin is closely correlated to impairment of endothelial function in healthy humans (21) and patients with diabetes (38). The effect of adiponectin on the endothelium is suggested to be mediated by its ability to stimulate NO (22). Disruption of the adiponectin gene causes disruption of IRS-1-associated PI 3-kinase activity, whereas viral-mediated reconstitution of adiponectin expression is able to reverse insulin resistance (39). ACE inhibition increases plasma levels of adiponectin in insulin-resistant subjects (19). One study has shown that adiponectin gene expression is not exclusively confined to adipose tissue but is also inducible in muscle cells (40). We therefore suggest that the increase in adiponectin gene expression in vascular tissue may contribute to the observed increase in insulin-stimulated endothelial function in our study because the increased availability of adiponectin may enhance endothelial nitric oxide synthase activation in the endothelium. We observed a 25-fold higher relative expression of adiponectin in adipose tissue than in the vascular tissue, yet no change was seen with quinapril. Because adipose tissue is the predominant source of circulating adiponectin, the change in vascular adiponectin gene expression in our study is therefore not suggested to affect the systemic adiponectin concentration, which was also confirmed. Of note, enhanced expression of adiponectin in adipose tissue has been correlated with an increased concentration of circulating adiponectin and improved systemic insulin action (25). Another possible mechanism for the effect of ACE inhibition could be inhibition of angiotensin II. Angiotensin II increases serine phosphorylation of the insulin receptor and could impair insulin’s effect on endothelial nitric oxide synthase. This mechanism may collaborate the beneficial effects of ACE inhibition on insulin-stimulated endothelial function in our study and whole-body insulin sensitivity shown by other groups (12, 13). In conclusion, this study demonstrates that quinapril increases endothelial function and insulin-stimulated endothelial function without changing insulin-stimulated muscle glucose uptake in patients with type 2 diabetes. These observations, in the absence of effects on systemic insulin action, indicate tissue-specific insulin sensitizing actions of quinapril. Acknowledgments Received June 1, 2005. Accepted December 1, 2005. Address all correspondence and requests for reprints to: Dr. Thomas S. Hermann, Bispebjerg University Hospital, Y-forskning, bygning 40, Bispebjerg Bakke 23, 2400 Copenhagen, Denmark. E-mail: [email protected]. This work was supported by The Danish Heart Foundation and the Danish Diabetes Association.


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