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Department of Clinical Pharmacology-TARGET (B.J., S.D., N.H., H.-G.E.) and Department of Medical and Chemical Laboratory Diagnostics (O.F.W.), Vienna University Hospital School of Medicine, A-1090 Wien, Vienna, Austria; and Department of Clinical Chemistry and Pathobiochemistry, University of Leipzig (V.R.), Leipzig, Germany
Address all correspondence and requests for reprints to: Bernd Jilma, M.D., Department of Clinical Pharmacology-TARGET, The Adhesion Research Group Elaborating Therapeutics, Vienna University Hospital School of Medicine, Währinger Gürtel 18-20, A-1090 Wien, Austria. E-mail: bernd.jilma{at}univie.ac.at
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Abstract |
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The study design was balanced, randomized, placebo-controlled, double blind, and cross-over. Twelve healthy male subjects received, on separate study days, a euglycemic hyperinsulinemic clamp (3 mU/kg·min) or placebo over 6 h. Plasma levels of cICAM-1, vascular cell adhesion molecule-1, circulating E-selectin, and sTM were measured by enzyme immunoassay; vWF-Ag was measured using a STA clot analyzer. Plasma levels of these adhesion molecules and endothelial cell activation markers were not affected despite a 30-fold increase in insulin levels.
Hyperinsulinemia has no adverse effect on circulating ICAM-1, vascular cell adhesion molecule-1, E-selectin, vWF, or sTM and therefore does not directly induce endothelial activation.
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Introduction |
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The exact mechanisms leading to increased cAM levels in DM, however, remain ill defined (3, 8), although several factors have been implicated: genetic inheritance (6) macro- and microvascular disease (8, 9), oxidative stress (10, 11), advanced glycation end products (12), hyperglycemia (13), insulin resistance (14), and hyperinsulinemia (15). Yet, except for one recent publication that examined VCAM-1 regulation by hyperinsulinemia (16), no studies have directly addressed the question of whether insulin increases cAM levels or other endothelial markers in vivo. This is of interest because cICAM-1 has been suggested as a cardiovascular risk factor (17), and the role of hyperinsulinemia as an independent risk factor remains controversial (18).
To examine effects of hyperinsulinemia on cAM levels, we measured the effects of a hyperinsulinemic euglycemic clamp on cAM levels. In addition, we quantified plasma levels of two other endothelial markers, von Willebrand factor (vWF) and soluble thrombomodulin (sTM), which are elevated in diabetes (2, 3, 19).
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Subjects and Methods |
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After approval of the study protocol by the ethics committee of the Vienna University School of Medicine, written informed consent was obtained from 12 healthy male subjects, aged 27 ± 3 yr (mean ± SD). To limit exposure of healthy volunteers to the stressful procedure, we designed the study to concomitantly assess the influence of hyperinsulinemia on ocular blood flow, results of which are considered for publication elsewhere. All volunteers passed a prestudy screening during the 4 weeks before the first study day, which included a physical examination, medical history, a standardized oral glucose tolerance test (oGTT), and a battery of laboratory tests as described previously (20).
Study design
The study design was randomized, placebo controlled, double blind, balanced, three-way cross-over with a washout period of at least 5 days.
Description of study days
Subjects were studied after an overnight fast. After an initial resting period of 45 min the euglycemic clamps were performed as previously described (21). All subjects received on different study days infusions of the endothelin antagonist BQ123 at the end of a placebo (saline) clamp or euglycemic hyperinsulinemia or received placebo at the end of euglycemic hyperinsulinemia. BQ123 was infused for studying its effects on ocular hemodynamics. Each clamp was started with a primed infusion of insulin for 6 min followed by a constant infusion rate of insulin at 3 mU/kg·min over 6.5 h with a concomitant infusion of BQ-123 (dose, 60 µg/min) or placebo over 30 min, commencing 6 h after the start of the insulin infusion. Therefore, it was guaranteed that the 6-h blood sampling was unaffected by BQ123. As a 6-h endothelin-1 infusion had no effect on cAM levels over a 24-h observation period (20), we assumed that a 30-min BQ123 infusion would not have any major effect on cAM levels at 24 h either.
Potassium chloride was infused at a rate of 150 mL/h to prevent hypokalemia. Glucose was infused at a rate necessary to maintain a constant blood glucose level of approximately 5.5 mmol/L. Arterialized venous blood samples were drawn for measurement of glucose concentration every 5 min from the contralateral arm placed in a heating blanket. All infusions were administered using an automatic device.
Rationale for doses
A euglycemic insulin clamp of 1.5 mU/kg·min increased insulin levels approximately 10-fold and caused a 10% increase in renal plasma flow (21). In the present study a dose of 3 mU/kg·min insulin was selected to cause a clear-cut insulin-mediated effect over a prolonged period to maximize the likelihood of detecting an effect on cAM regulation.
Methods
All parameters were measured from citrated plasma. Plasma was separated by immediate centrifugation for 15 min at 2000 x g and was kept frozen at -80 C until analysis in duplicate. All cAM were assayed with commercially available EIA purchased from R & D Systems (Oxon, UK), sTM with an enzyme immunoassay from Diagnostica Stago (Chausson, Asnieres-sur-Seine, France) as previously described (22). vWF antigen (vWF-Ag) was determined on a STA Clot analyzer (STA Liatest vWF, Roche Molecular Biochemicals, Mannheim, Germany). Serum insulin concentrations were determined by RIA (Seroneo, Freiburg, Germany).
An N Latex CRP mono particle-enhanced immunonephelometric assay (Dade Behring, Marburg, Germany) was used to determine C-reactive protein from serum samples. Intra- and interassay coefficients of variation were less than 5% and 6%, respectively. The lower limit of sensitivity is 0.175 mg/L.
Sample size estimation and data analysis
An a priori sample size calculation was based on the intrasubject variability of cAM levels (5–8%). According to clinical studies (7, 9, 17, 23) detection of a 20% increase in cAM after insulin infusion was considered to be clinically relevant. A sample size calculation (24) indicated that nine subjects would be sufficient to detect such an increase in cAM if it occurred after insulin infusion.
Data are presented as the percent change from baseline [mean ± 95% confidence intervals (CI)]. Plasma levels of all parameters were corrected for hemodilution using changes in albumin (mean change, 9–14% at 6 h of all periods) as a correction factor. All treatment effects were analyzed using the Friedman ANOVA and the Wilcoxon signed rank test for post-hoc analysis. The Spearman ranks correlation test was used to correlate baseline data. A two-tailed P < 0.05 was considered significant.
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Results |
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). All except one subject during the
insulin period exhibited normal CRP levels. Only after pooling of data
from all three periods were CRP levels found to correlate with sTM
(r = 0.44; P = 0.016), but not with the other
markers measured.
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Yet, insulin had no effect on cAM levels (Fig. 1). Similarly, insulin did not affect
plasma levels of sTM or vWF-Ag, although greater variation was observed
for vWF-Ag compared to the other parameters (Fig. 2). None of the parameters increased by
the preset level of 20%, which was considered clinically relevant.
Interestingly, cVCAM-1 increased vs. baseline in all
periods, but without significant differences between treatment periods.
This trend was also observed for vWF-Ag.
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Discussion |
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When confronted with negative results we have to ask whether the study design was adequate. First, our euglycemic insulin clamp infusion increased insulin concentrations 30-fold above control conditions. Thus, the calculated daily insulin dose of about 300 U was not likely to be too low, because it elevated insulin levels 10-fold above those concentrations seen in NIDDM patients with increased cAM levels (9, 25, 26). Secondly, the 6-h duration of the insulin infusion or the 24-h observation period may be regarded as short; however, an increase in cAM levels can be seen as early as 2 h after infusion of endotoxin, yielding 700% higher cE-selectin levels after only 4 h (22).
Thirdly, infusion of the endothelin receptor antagonist BQ123 from 6–6.5 h for ophthalmological investigations could not possibly affect levels measured at 6 h. Further, as endothelin-1 does not influence cAM levels (20), BQ123 is not likely to have any effects on cAM concentrations. This idea is also supported by the finding that insulin infusion with or without BQ123 yielded similar results.
Surprisingly, there was an increase in cVCAM-1 in all three treatment periods, and a similar trend was observed for vWF-Ag. As this effect was most pronounced during placebo infusion, this effect cannot be ascribed to insulin. Possibly the high water load led to glomerular hyperfiltration, a condition that can up-regulate ICAM-1 expression at least in animals (27). Alternatively, altered shear stresses may have affected cVCAM-1 levels.
Although we cannot rule out the possibility that insulin might differently affect cAM levels in diabetics, a hyperinsulinemic clamp leads to comparable serum insulin concentrations and insulin receptor kinase activity in NIDDM patients compared to controls (28).
In conclusion, our experiments do not support the concept that high circulating levels of cAM or other endothelial activation markers in diabetics are due to a direct or acute action of insulin. Hence, hyperinsulinemia does not directly induce endothelial activation, but as demonstrated recently, elevated cAM levels may be due in part to insulin resistance (14).
Received August 20, 1999.
Revised November 2, 1999.
Accepted December 3, 1999.
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This article has been cited by other articles:
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  C. Cardillo, M. Mettimano, N. Mores, K. K Koh, U. Campia, and J. A Panza Plasma levels of cell adhesion molecules during hyperinsulinemia and modulation of vasoactive mediators Vascular Medicine, August 1, 2004; 9(3): 185 - 188. [Abstract] [PDF]
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 G. Targher, R. C. Bonadonna, M. Alberiche, M. B. Zenere, M. Muggeo, and E. Bonora Relation Between Soluble Adhesion Molecules and Insulin Sensitivity in Type 2 Diabetic Individuals: Role of adipose tissue Diabetes Care, November 1, 2001; 24(11): 1961 - 1966. [Abstract] [Full Text] [PDF]
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 A. D. Blann and G. Y. H. Lip Editorial: Cell Adhesion Molecules in Cardiovascular Disease and Its Risk Factors--What Can Soluble Levels Tell Us? J. Clin. Endocrinol. Metab., May 1, 2000; 85(5): 1745 - 1747. [Full Text]
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