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The Differential Effects of Metformin on Markers of Endothelial Activation and Inflammation in Subjects with Impaired Glucose Tolerance: A Placebo-Controlled, Randomized Clinical Trial

Clinical Research Center (A.E.C.), Joslin Diabetes Center, Harvard Medical School, Boston, Massachusetts 02215; and Departments of Endocrinology and Metabolism (A.D., C.A.A.-S., A.N.H., J.L.C., F.J.G.-P.), Radiology (T.C.), and Medicine (J.A.R.), Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Tlalpan 14000, Mexico City, Mexico

Address all correspondence and requests for reprints to: Carlos A. Aguilar-Salinas, M.D., Department of Endocrinology and Metabolism, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Vasco de Quiroga Number 15, Tlalpan 14000, Mexico City, Mexico. E-mail: caguilarsalinas{at}yahoo.com.

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
The effect of metformin (1000 mg twice a day) on markers of endothelial activation, inflammation, and coagulation was investigated in subjects with impaired glucose tolerance (IGT) in a 16-wk, randomized, placebo-controlled, double-blind study. Soluble intercellular adhesion molecule, soluble vascular cell adhesion molecule, C-reactive protein, TNF, von Willebrand factor, and tissue plasminogen activator were measured at baseline and at the end of the trial. Subjects with IGT (n = 55, 14 males and 41 females), aged 48.4 ± 9.6 yr with a body mass index of 31.4 ± 5.6 kg/m², were studied. All participants followed a 1-month stabilization period in their diet and physical activity. Afterward, 29 subjects were assigned to the treatment group and 26 to the control group. A significant reduction in weight, fasting plasma glucose, soluble intercellular adhesion molecule (306 ± 75 vs. 268 ± 61 ng/ml, P = 0.029), soluble vascular cell adhesion molecule (595 ± 114 vs. 508 ± 126 ng/ml, P = 0.006), and von Willebrand factor (124 ± 34 vs. 94 ± 34%, P = 0.001) was seen in the treatment group, whereas tissue plasminogen activator, TNF, and C-reactive protein levels did not change. No change was seen in the control group. Thus, metformin improves the plasma levels of some markers of endothelial activation and coagulation in subjects with IGT, whereas it has no effect on markers of inflammation.

	Introduction

Top Abstract Introduction Subjects and Methods Results Discussion References

 
IMPAIRED GLUCOSE TOLERANCE (IGT) represents a state that not only increases the risk for type 2 diabetes but also for cardiovascular disease (1). Endothelial dysfunction and low-grade inflammation are important abnormalities in people with IGT that may contribute to this dual risk (2, 3, 4). The Diabetes Prevention Program demonstrated the feasibility of reducing the risk of progression to type 2 diabetes in people with IGT through lifestyle modification or the use of metformin (5, 6). Metformin has a beneficial effect on traditional cardiovascular risk factors (7) and is associated with decreased cardiovascular morbidity and mortality in people with type 2 diabetes (8, 9). In addition, metformin use has been associated with a favorable effect on C-reactive protein (CRP), plasminogen-activator inhibitor 1 (PAI-1) levels, and vascular reactivity in diabetic subjects (10, 11, 12, 13). Some of these beneficial effects on markers of inflammation and coagulation may be related to improved glycemia. It is uncertain whether these findings are fully applicable to subjects with IGT and whether these beneficial effects are the result of a direct effect of the medication on vascular function or its frequently associated weight loss and reduction of hyperglycemia. The Biguanides and the Prevention of the Risk of Obesity-1 study (14), conducted in overweight individuals, showed an improvement on tissue plasminogen activator (tPA), PAI-1, and von Willebrand factor (vWF) levels. Unfortunately, studies required for the diagnosis of IGT were not done. Thus, our objective was to assess the effect of metformin on a wide range of markers of inflammation (i.e. CRP and TNF), endothelial activation [i.e. soluble intercellular adhesion molecule (sICAM) and soluble vascular cell adhesion molecule (sVCAM)], and coagulation (vWF and tPA) in people with IGT in a randomized, placebo-controlled, double-blind clinical trial.

	Subjects and Methods

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Subjects

A total of 55 subjects with IGT, 25–65 yr of age, were included in the study. The participants were identified at the outpatient diabetes clinic of the Instituto Nacional de Ciencias Médicas y Nutrición (INCMN). Most study volunteers were relatives of patients that attend this clinic for diabetes care. IGT was diagnosed using the World Health Organization definition for the oral glucose tolerance test (15). To avoid confounding factors known to affect plasma markers of endothelial function, coagulation, and/or inflammation, the following exclusion criteria were applied to all subjects: smoking any amount of cigarettes during the previous 6 months, cardiac arrhythmia, congestive heart failure, recent stroke, chronic renal disease, macroalbuminuria (expressed as albumin to creatinine ratio > 300 µg/mg), severe dyslipidemia [triglycerides > 600 mg/dl (6.74 mmol/l) or cholesterol > 300 mg/dl (7.89 mmol/liter)], any severe chronic disease, or any acute or chronic inflammatory illness. Subjects were also excluded if they were on any medications with known effects on glucose or lipoprotein metabolism. In addition, subjects were excluded from the study if they exhibited any contraindication for the use of metformin. The ethics committee approved the protocol, and all participants gave written informed consent.

Protocol

Patients attended the outpatient diabetes clinic of the INCMN for a screening visit in which a detailed medical history and a general physical examination were conducted by a study physician. A standard 75-g oral glucose tolerance test was performed. Subjects’ weight, height, blood pressure, and body mass index (BMI) were also obtained. All blood samples for the measurement of various general, metabolic, and vascular factors (described below) were obtained after an overnight fast and a 24-h period of abstinence from alcohol and vigorous exercise. Eligible subjects with IGT were included in the study and randomized to receive metformin or placebo. A random allocation sequence was generated before the beginning of the study; blocks of 10 patients were incorporated to assure a similar number of participants in each treatment group. Numbered containers were used. The containers of the excluded cases were discarded to keep the randomization sequence as originally prepared. Participants in both groups received placebo for the initial 4 wk to assess their compliance with the study medication. During this run-in phase and throughout the rest of the study, patients were asked to follow an isocaloric diet composed of 50% carbohydrates, 20% protein, 30% fat, and 30 g/d fiber and were asked not to modify their usual physical activity. At the end of the first 4 wk, eligible participants, defined as those whose adherence to the medication was greater than 80% through the pill-counting method and whose body weight had not changed by more than 3 kg, were allowed to continue to receive treatment with metformin or placebo for the following 16 wk. During the 1st month of active treatment, participants received 1 g metformin or matching placebo, and the dosage was increased and maintained at 2 g/d (1000 mg twice a day) thereafter. The participants were seen every 4 wk for the duration of the study that included 4 wk of the stabilization period and 16 wk of active treatment. At every visit, a clinical and safety evaluation was performed, and adherence to treatment was assessed. At the end of the study period, all initial evaluations were repeated. This study was conducted as a randomized, placebo-controlled, double-blind clinical trial. The sponsor generated the allocation sequence; numbered containers were prepared for every visit. The placebo and metformin tablets had the same shape and color. Researchers were blinded throughout the study. The allocation sequence was not revealed until all the evaluations were completed.

Efficacy parameters

The efficacy evaluation endpoints were the percent change from baseline to the end of the active treatment period (16 wk) for sICAM, sVCAM, vWF, CRP, TNF, and tPA. The baseline value for all these measurements was obtained after the 4-wk run-in period when people in the treatment group started taking the active medication. Secondary efficacy evaluations were the percent change from baseline to the end of active treatment period for insulin, total cholesterol, triglycerides, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, non-HDL cholesterol, fasting plasma glucose (FPG), and hemoglobin A_1C (A1c) levels. Insulin sensitivity was estimated using the homeostasis model of assessment (HOMA) score (16). With this method, high HOMA scores denote low insulin sensitivity.

Safety evaluation

Before participants were randomized in the study, a complete physical examination and a clinical laboratory evaluation were performed. The laboratory evaluation included a blood cell count, pregnancy test, urinalysis, liver function tests, and a glycemic profile (FPG and A1c). All these tests were repeated at the end of the study. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), and FPG were measured at all study visits. If ALT and/or AST values were above the upper limit of normal, a second measurement was performed within 1 wk. If either the ALT or AST concentrations were above 3 times the upper limit of normal at any point during the study, the participant was asked to stop the study medication, and standard safety measures were implemented.

Laboratory evaluations

The Department of Endocrinology and Metabolism central laboratory of the INCMN performed the lipid and clinical laboratory measurements using standardized procedures. This laboratory is certified for standardization of the tests by the External Comparative Evaluation of Laboratories Program of the College of American Pathologists. The markers of inflammation and endothelial activation were measured at the Clinical Research Center of the Joslin Diabetes Center (Boston, MA). FPG, total cholesterol, HDL cholesterol, triglycerides, liver function tests, and creatinine were measured using the Synchron CX analyzer (Beckman Systems, Fullerton, CA). The coefficients of variation for cholesterol and HDL cholesterol are 3.3 and 2.5%, respectively. LDL cholesterol concentration was estimated by the Friedewald formula (17). Plasma insulin concentrations were estimated using a RIA method. Glycosylated hemoglobin was determined in whole blood using ion-exchange HPLC (normal range, 4–6%). vWF (Asserachrom, American Bioproducts, Parsippany, NJ), sVCAM, sICAM, TNF (R&D Systems, Minneapolis, MN), tPA antigen (IMUBIND, American Diagnostics), and high-sensitivity CRP (Diagnostics Products Corp., Los Angeles, CA) were measured in plasma in duplicate using an ELISA method.

Statistical analysis

Statistical analysis was performed using SPSS for Windows version 10 (SPSS Inc., Chicago, IL). The sample size allows for the detection of a difference of 6% or greater in any of the primary efficacy outcome measurements (with an -error of 0.05 and a ß-error of 0.1). An intention to treat analysis was used. Two-sided ANOVA tests were used for assessing differences between groups for continuous variables. All categorical variables were analyzed using the ² test. Spearman correlation coefficients were used to assess association between variables. Multiple logistic regression models were constructed for the identification of variables associated with the concentration of markers of inflammation (i.e. CRP and TNF), endothelial activation (i.e. sICAM and sVCAM), and coagulation (vWF and tPA) before and after treatment.

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Baseline characteristics of the participants

The participant flow is described in Fig. 1. A total of 60 eligible subjects with IGT were identified during the screening process (completed in a 1-yr period). All of them were eligible to continue in the study based on their compliance with run-in placebo and adherence to a stable diet; however, five of them refused to continue in the study because they could not comply with the scheduled visits. A total of 29 individuals were randomized to the metformin group and 26 to the control group. Table 1 shows the baseline characteristics of the intervention and the control groups. There were no statistically significant differences in any of the study variables at baseline between groups.

View larger version (24K): [in this window] [in a new window]   FIG. 1. Flow diagram of the disposition of patients through the phases of the study.

Data in 90% of the patients were available for final statistical assessment that was conducted as an intention to treat analysis. Table 1 shows the effect of metformin and placebo on the metabolic variables and cardiovascular risk factors assessed in the study. There was a statistically significant reduction in weight and BMI in both the intervention and the control groups. The mean reduction in weight in both groups was very similar. The mean weight change in the metformin group was 2.4 kg, corresponding to a weight loss of 3%, and 1.7 kg in the placebo group, corresponding to a weight loss of 2.2%. There was no statistically significant difference in the mean body weight between the groups at any point in time during the study. A mean reduction of 1 kg (41.6% of total weight loss) in the metformin group was achieved during the run-in period; therefore, it cannot be attributed to the medication. A mean reduction of 0.4 kg (23.5% of total weight loss) in the placebo group was achieved during this initial period. The mean FPG level significantly dropped in both groups as well. However, no statistically significant differences in FPG were appreciated between the groups at any point in time during the study period. No statistically significant changes were appreciated in blood pressure, A1c, HOMA values, and lipids in response to the study treatment in any of the two groups.

Markers of endothelial activation, coagulation, and inflammation

The main results on the plasma markers of endothelial activation, coagulation, and inflammation are shown in Table 2.

Values in response to metformin or placebo. Table 2 shows the mean values of the various plasma markers of endothelial activation, coagulation, and inflammation in response to the assigned intervention. A statistically significant reduction in the plasma values of sICAM, sVCAM, and vWF was seen in the metformin group whereas no statistically significant change was seen in the plasma values of tPA, CRP, and TNF. No statistically significant changes were seen in any of these markers in the placebo group.

To assess in more detail how the various study variables contributed to the appreciated changes in the plasma values of the markers of endothelial activation, coagulation, and inflammation, we conducted further statistical analysis. The change (delta) in vWF levels from baseline had a significant correlation with the change (delta) from baseline in sVCAM levels (r = 0.29, P = 0.04). Other significant correlations were delta in vWF and delta in HOMA (r = 0.44, P = 0.02) and delta in vCAM and delta in weight (r = 0.38, P = 0.04). In a multiple regression model (r² = 0.687, P < 0.001), after controlling for possible confounding variables (including changes in weight, HOMA, plasma lipids, and blood pressure), the effect of metformin on vWF levels remained statistically significant (ß-coefficient = 27.49 ± 6.9, P < 0.001). The same phenomenon was observed for the sVCAM and sICAM concentrations. These results suggest that the effect of metformin treatment on plasma levels of sVCAM, sICAM, and vWF levels is independent from its effects on weight and insulin sensitivity.

Follow-up and side effects

Compliance with the study medications was above 90% in both groups. No participants had to be excluded from the study due to elevation of liver enzymes or any serious adverse reaction to the study medication.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Metformin significantly reduces the plasma levels of some markers of endothelial activation and coagulation such as sVCAM, sICAM, and vWF in subjects with IGT, whereas it has no effect on other markers of subclinical inflammation such as TNF and CRP. The beneficial effects on the adhesion molecules and vWF levels are not explained by weight loss and/or improved insulin sensitivity. The data reported here provide new mechanistic insights regarding the vascular protective effects of metformin (9). Additional studies, based on clinical outcomes, are needed to confirm the beneficial effects of metformin on the atherosclerosis process and for assessing the contribution of the changes reported here to the protective actions of the drug.

The finding that metformin has these differential effects on some markers of endothelial function in people with IGT is interesting and may have some important clinical implications. Cellular adhesion molecules are expressed on endothelial cells in response to inflammation and facilitate the adhesion of circulating leukocytes to their surface (18). The soluble forms of these molecules are found in the circulation, and increased levels have been associated with increased risk of coronary events (19, 20, 21). The adhesive protein vWF contributes to platelet function by mediating the initiation and progression of thrombus formation at sites of vascular injury (22), whereas tPA is an important component of the fibrinolytic system (23).

The beneficial effect of metformin on the markers of endothelial activation may result either from direct effects on endothelial cells and/or indirect actions (i.e. by decreasing IR, promoting weight loss, and lowering the plasma levels of toxic compounds to the endothelium such as glucose and free fatty acids). In the present study, a significant correlation was found between the change in vWF and HOMA values in the study population as a whole (r = 0.44, P = 0.02). However, despite similar changes in glycemia and weight in the treatment and placebo groups, only those subjects exposed to metformin exhibited a reduction in the plasma levels of sVCAM, sICAM, and vWF. Furthermore, in a multiple regression model, after controlling for possible confounding variables (including changes in weight, blood pressure, HOMA, plasma glucose, and lipids), the effect of metformin on these markers of endothelial activation remained statistically significant. These findings suggest that metformin may have a direct beneficial effect on vascular cells in vivo. Metformin reduces the alloxan-induced hyperpermeability in normoglycemic hamsters and protects against the loss of vasomotion caused by hyperglycemia in bats (24, 25). It also reduces oxidative stress (26, 27). More recently, metformin has been shown to inhibit monocyte adhesion to human endothelial cells and foam cell formation (28). These studies are in agreement with our findings suggesting that metformin has direct effects on vascular function. The precise mechanisms for any direct action of metformin on the vasculature are unknown. However, metformin increases the activity of AMP-K, a stress-activated protein kinase that participates in the regulation of energy and metabolic homeostasis (29, 30, 31). This enzyme is located in multiple tissues, including the vascular endothelium. Its activity in endothelial cells is stimulated by adiponectin and exercise, factors known to improve endothelial function (32, 33). Whether AMP-K mediates the direct beneficial effects of metformin on vascular cells deserves further investigation.

Metformin did not have any effect on TNF and CRP levels in our study population. These are considered markers of subclinical inflammation with a clear connection to IR (34, 35, 36, 37, 38, 39, 40). Metformin has been shown to reduce CRP levels in people with type 2 diabetes and women with polycystic ovary syndrome (10, 41, 42). However, it is uncertain whether these beneficial changes are due to a direct effect or the result of improved glycemia, insulin sensitivity, and weight. Hyperglycemia is known to affect the levels of inflammatory markers (43). In addition, weight loss and improved insulin sensitivity can improve endothelial function and reduce the plasma levels of some markers of endothelial activation (44). The Biguanides and the Prevention of the Risk of Obesity-1 study found that the beneficial effect of metformin on PAI-1 levels was only seen in those individuals who lost weight, whereas the effect on vWF was independent of weight change (14). Our study design allowed us to have a more accurate estimation of the direct effects of metformin because a run-in period with diet and placebo was included. These factors were not controlled in previous reports; therefore, the direct beneficial effects of metformin may have been overestimated. The subjects in the intervention group in our study lost a mean of 1.75% of their body weight after exposure to metformin; therefore, this weight loss may have been insufficient to observe a significant reduction in the markers of inflammation. Our study included a larger number of individuals in comparison with other reports that showed a reduction in CRP values. Hence, the lack of change in CRP is not explained by an inadequate statistical power in our study. In addition, the dosage of metformin and the duration of our study assured the detection of the effects of metformin on the various markers of inflammation. Our findings support the concept that metformin has a weak direct antiinflammatory effect and that its impact on inflammatory markers is likely to be the result of improved glycemia and weight.

In conclusion, metformin has a differential effect on the plasma levels of markers of endothelial activation and inflammation in subjects with IGT. It reduces the levels of sVCAM, sICAM, and vWF but has no effect on the levels of CRP and TNF. The beneficial effects on the markers of endothelial activation are not related to changes in glycemia, lipids, weight, or insulin sensitivity in our study population. These findings suggest that metformin may have direct actions on vascular cells. Elucidating the precise mechanisms for these direct effects deserves further investigation. The overall clinical impact of metformin on cardiovascular outcomes in people with IGT remains to be determined.

	Footnotes

 
This work was supported by a grant from Silanes Laboratories through FunSalud, a nonprofit health care organization in Mexico.

Abbreviations: A1c, Hemoglobin A_1C; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BMI, body mass index; CRP, C-reactive protein; FPG, fasting plasma glucose; HDL, high-density lipoprotein; HOMA, homeostasis model of assessment; IGT, impaired glucose tolerance; IR, insulin resistance; LDL, low-density lipoprotein; PAI-1, plasminogen-activator inhibitor 1; sICAM, soluble intercellular adhesion molecule; sVCAM, soluble vascular cell adhesion molecule; tPA, tissue plasminogen activator; vWF, von Willebrand factor.

Received January 6, 2004.

Accepted April 7, 2004.

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