Introduction

Adiponectin is an adipocyte-specific hormone that is abundantly expressed and secreted so that it reaches high concentrations in the human blood (5 to 20 g/mL) (1, 2) . The blood levels of adiponectin are reduced in obesity and type 2 diabetes (T2D),¹ and hypoadiponectinemia is associated with the development of T2D in Pima Indians (3, 4) . Adiponectin has various metabolic actions, including an improvement of glucose uptake by the skeletal muscle cells and a reduction of hepatic glucose production (5, 6) . Furthermore, adiponectin has anti-atherogenic and anti-inflammatory properties. Thus, in animal models, adiponectin can prevent neointimal thickening after artery injury and attenuate DNA synthesis induced by various growth factors such as platelet-derived growth factor, epidermal growth factor, and basic fibroblast growth factor (7). In addition, adiponectin increases interleukin-10 expression and reduces nuclear factor- signaling, endothelial adhesion molecule expression, transformation from macrophage to foam cell, and tumor necrosis factor expression (8).

Increased production of advanced glycation end-products (AGEs) is another mechanism that is related to the development of cardiovascular disease in diabetes. More specifically, AGEs exert a positive feedback on the expression of the receptor for AGEs (RAGE) and cause nuclear factor- activation that leads to inflammation and increased vascular cell adhesion molecule (VCAM) formation (9, 10). Chronic overexpression of RAGE results in vascular smooth muscle cell proliferation and neointimal expansion that eventually leads to progression of atherosclerosis, plaque instability, and the emergence of clinical events (11).

Thiazolidinediones are a new class of compounds that improve insulin resistance and are extensively used in the treatment of T2D (12). They exert their action by binding the peroxisome proliferator activator receptor (PPAR)-, a nuclear receptor that is located mainly in adipocytes, but that is also expressed in skeletal muscle, macrophages, and the vasculature (13). In the adipose tissue, activation of the PPAR- receptors promotes differentiation, a mechanism that has been related to the improvement of insulin resistance (14). Because thiazolidinediones have also been shown to significantly increase the synthesis and secretion of adiponectin by adipocytes, it has been suggested that the modulation of the adiponectin plasma levels may play an important role in the reversal of insulin resistance (15, 16).

In addition to their effects on insulin resistance, thiazolidinediones exert a variety of additional beneficial effects, including a decrease in blood pressure, improvement of diabetic dyslipidemia and fibrinolysis, and a decrease in the platelet aggregation and vascular smooth-muscle proliferation (17). In addition, a recent study in our unit has indicated a beneficial effect of troglitazone, the first approved thiazolidinedione, on the endothelial function of patients with recently diagnosed T2D and without any serious long-term complications (18). In this study, we examined whether there is a relationship between the changes in adiponectin plasma levels and the changes in endothelial function in the subjects who participated in the above study. In addition, we have examined the effect of troglitazone on RAGE expression in the same population.

Research Methods and Procedures

Subjects

Seventy-two T2D patients were recruited and completed the study. More details about the recruited subjects have been provided elsewhere (18). In brief, the main inclusion criteria were T2D patients 21 to 70 years of age. The main exclusion criteria were smoking during the previous 6 months, cardiac arrhythmia, congestive heart failure, recent stroke, chronic renal disease, macroalbuminuria (expressed as albumin/creatinine ratio > 300 g/mg), severe dyslipidemia (triglycerides > 600 mg/dL or cholesterol > 300 mg/dL), chronic disease requiring active treatment, and treatment with glucocorticoids, antineoplastic agents, psychoactive agents, bronchodilators, or any thiazolidinedione. Because of the known potential hepatotoxicity of the study drug, a particularly careful review of this effect was performed with all participants, and only individuals without any history of liver disease and with completely normal liver function tests, including liver enzymes, were allowed to participate in the study. Seven patients were treated with -blockers, 5 with angiotensin-converting enzyme inhibitors, 14 with a combination of these two medications, and 30 with aspirin. Fourteen patients were on diet alone, 55 were on oral hypoglycemic agents, and 7 were on insulin. There were no differences between the active and placebo groups.

The protocol was approved by the ethics committee or institutional review board at each center, and all participants gave written informed consent.

Methods

All participants were seen at the Joslin Diabetes Center and the Microcirculation Laboratory at the Beth Israel Deaconess Medical Center. All patients initially underwent a physical examination, blood drawing, and measurement of the vascular reactivity of the macro- and microcirculation previously described (18). In brief, high-resolution ultrasound images were used to measure the flow-mediated dilation (FMD, endothelium-dependent) and nitroglycerin-induced dilation (NID, endothelium-independent) in the brachial artery. All images were obtained by two specifically trained sonographers who worked exclusively in the vascular laboratory. The data were analyzed by a radiologist (R. Saouaf, director of the vascular laboratory) with particular interest in vascular reactivity measurements. The coefficient of variation for both FMD and NID measurements was <5% (18). Laser Doppler perfusion imaging was employed to measure vasodilation in the forearm skin microcirculation in response to iontophoresis of 1% acetylcholine (endothelium-dependent) and 1% sodium nitroprusside (endothelium-independent). The coefficient of variation of the baseline measurement was 14.1% and during maximal hyperemic response after the iontophoresis was 13.7% (18).

The participants were randomized to treatment with either 600 mg of troglitazone or placebo for a 12-week period. At the end of the treatment period, patients were seen again and repeated all tests that had been done during the pretreatment visit. Serum adiponectin levels were measured using an adiponectin ELISA according to the manufacturer's specifications (B-Bridge International, Sunnyvale, CA). The measurement of the biochemical markers of endothelial dysfunction was performed soon after the completion of the study. Adiponectin was measured 12 to 18 months after the study completion. There is no indication that the extended storage of samples has an effect on adiponectin measurements. In this study, this was confirmed by the clear difference that was observed in the increase of the adiponectin levels between the active and placebo group.

Skin Biopsies

Twenty-one subjects agreed to participate in the skin biopsy part of the protocol. There were no differences in the baseline characteristics between those who participated and those who did not participate in this part of the study.

The skin biopsies were performed the same day that vascular reactivity measurements were performed, and blood specimens were taken for the measurement of biochemical markers of endothelial dysfunction. Two 2-mm skin punch biopsies were taken from the volar aspect of the forearm under local anesthesia (1% plain lidocaine). The skin wound edges were approximated with sterile strips, and no sutures were used. Biopsies were immediately frozen in liquid nitrogen and stored at -70 °C.

RAGE, PPAR, and CD31 expression were determined by Western blot. In brief, tissue biopsies were homogenized with individual 1-mL Micro Duall tissue grinders (Kimble Konte, Vineland, NJ) in extraction buffer containing 1% sodium dodecyl sulphate (Sigma, St. Louis, MO), 1 mM sodium vanadate (Sigma), and 50 mM Tris HCl, pH 7.4 (Sigma). Protein extracts were obtained by centrifugation of the lysate at 4 °C, and protein concentrations were determined using a detergent compatible protein assay kit according to the manufacturer (Bio-Rad, Hercules, CA). Protein (40 g) was separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane (Millipore, Bedford, MA) using Bio-Rad Trans-Blot Cell. Membranes were blocked for 30 minutes in TBS/casein (Bio-Rad) and incubated with monoclonal antibodies directed against human CD31 (Zymed, San Francisco, CA) or PPAR (Santa Cruz Biotechnology, Santa Cruz, CA) at a 1/1000 dilution. For RAGE detection, a polyclonal rabbit antihuman RAGE IgG antibody (Santa Cruz Biotechnology) was used at a 1/1000 dilution. After washing in TBS, membranes were incubated with horseradish-conjugated antibodies (BD Transduction, San Diego, CA) and washed again. Membranes were incubated with species-appropriate horseradish-conjugated secondary polyclonal antibodies and washed again. Antigen detection was performed with a chemiluminescent detection system (Amersham, Piscataway, NJ). Resultant blots were digitized, and individual sample protein expression was determined by densitometry using NIH Image Software (NIH, Bethesda, MD). Protein expression was normalized to CD31 levels to reflect the microvascular content of RAGE and PPAR in each biopsy.

Data Analysis

The Minitab statistical package (Minitab, State College, PA) and the Statistical Analysis System (SAS, Cary, NC) were used for statistical analysis. The primary analysis examined was the difference in mean change between the placebo- and troglitazone-treated groups. The study was designed to detect a 50% improvement in the vascular reactivity measurements at a level of 0.80 and an level of 0.05 in each group separately. A two-tailed comparison was assumed with a significance level of p < 0.05. The distribution of the variables was tested for normality using Kolmogorov-Smirnov tests. Variables that did not meet the criteria for normality under these tests were analyzed with non-parametric methods (Kruskal-Wallis two-sample test) and are presented as median (25th to 75th percentile).

The change between baseline and exit visit in each group was evaluated using the paired Student's t test for parametrically distributed data and the Wilcoxon matched pair signed-rank test for non-parametrically distributed data. The Student's t test was used to compare the baseline characteristics between active and placebo groups. Correlation among variables was tested using both univariate and multivariate analyses (Pearson correlation and Spearman correlation analysis for parametrically and non-parametrically distributed data and analysis and multiple stepwise regression analysis, respectively). The results are presented as mean SD or median (25th to 75th percentile).

Results

The baseline characteristics of the active and placebo groups are shown in Table 1. No differences were observed between the two groups in any of the measured variables. Also, no differences were also observed in the baseline measurements of vascular reactivity, markers of endothelial activation, adiponectin, and RAGE levels (Table 2). When all patients were considered as one group, adiponectin levels correlated significantly with age (r = 0.26, p = 0.027), fasting plasma insulin (r = –0.023, p = 0.06), aspartate aminotransferase (r = -0.33, p = 0.005), and alanine aminotransferase (ALT) (r = -0.36, p = 0.002). However, no significant correlations were observed between adiponectin levels and any measurement of vascular reactivity at both the micro- and macrocirculation. Regression analysis showed that all these variables, with the exception of ALT, were independent predictors and accounted collectively for 29% of the observed variation in the adiponectin levels. A weak correlation was observed between FMD and systolic blood pressure (r = -0.29, p = 0.02). No correlations were observed between FMD and age or diabetes duration. Furthermore, when all participants were divided into two groups (those above the median age and those below the median age), no differences were noticed in FMD. No differences were also observed when participants were divided into those above and those below the median diabetes duration.

Table 1. - Baseline characteristics.

Full table

Table 2. - Baseline measurements of vascular reactivity and biochemical markers of endothelial function.

Full table

The changes between the exit and baseline visits in all measured variables are shown in Table 3. As would be expected, a significant reduction was noticed in the fasting plasma glucose and the hemoglobin A_1c (HbA_1c) of the active group compared with the placebo-treated patients (p < 0.0001). In addition, a significant increase was noticed in the total cholesterol of the active group compared with the controls (p = 0.01). The only other difference was in the adiponectin levels, where an average 75% increase was observed in the active group, whereas no changes were observed in the control group (p < 0.0001; Figure 1). No differences were found in the FMD and NID, the acetylcholine response, and the sodium nitroprusside response between the active and placebo groups (Table 3).

Figure 1.

: Changes in circulating adiponectin concentrations in the troglitazone- and placebo-treated T2D patients. Troglitazone-treated patients had a significant increase in adiponectin levels at the exit visit (gray column) compared with baseline (black column; *p < 0.001). In contrast, no changes were observed in the placebo group.

Full figure and legend (85K)

Table 3. - Changes between the exit and baseline visits in glycemic control, lipids, vascular reactivity, and other markers of endothelial function during the study.

Full table

The correlation between the changes in adiponectin levels and other measurements of glycemic control, lipid levels, vascular reactivity, and markers of endothelial dysfunction, when all participants were considered as one group, are shown in Table 4. An inverse correlation was noticed between the change in adiponectin and the change in fasting plasma glucose (r = -0.29, p < 0.05) and HbA_1c (r = -0.30, p < 0.05). Significant correlations were also found between the change in adiponectin and total cholesterol (r = 0.25, p < 0.05) and low-density lipoprotein (LDL)-cholesterol (r = 0.34, p < 0.01), whereas a marginal inverse correlation was observed with high-density lipoprotein (HDL)-cholesterol (r = -0.34, p = 0.07). However, no significant correlations were found between adiponectin level changes and changes in the measurements of vascular reactivity and markers of endothelial activation (Figure 2). Regression analysis showed that the changes in HbA_1c, total cholesterol, and HDL-cholesterol were independent predictors of adiponectin changes but could collectively account for only 25% of the observed variability.

Figure 2.

: Plot of the changes in adiponectin and the endothelium-dependent vasodilation of the brachial artery (FMD) in the active group that was treated with troglitazone. No correlation was observed between the changes in these two measurements.

Full figure and legend (54K)

Table 4. - Correlation between changes in adiponectin and other measurements during the 12-week period study.

Full table

Similar results were observed regarding the changes in adiponectin levels when the study patients were divided into three subgroups as they were divided in the previously reported results (18). Thus, the three groups were comprised of 1) patients with recently diagnosed diabetes, 2) patients with diabetes of long duration but no serious complications, and 3) patients with history of macrovascular disease. In the troglitazone-treated subgroup with recently diagnosed diabetes, the adiponectin change was 71 122% (percent increase over baseline); in the subgroup with long duration, it was 98 118%, and in the subgroup with macrovascular disease, it was 60 52% (p = not significant). The respective changes in the placebo-treated subgroups were -15 23%, (percent change from baseline), -4 26%, and 6 14% (p = not significant).

No changes were observed in RAGE levels in response to treatment with troglitazone (Figure 3). In addition, no correlations were found between changes in RAGE levels and any other measurement of glycemic control or endothelial function. At baseline, a strong correlation was found between PPAR and RAGE levels (r = 0.64, p < 0.05). No associations were found between PPAR levels and any other measurement.

Figure 3.

: Changes in the expression of RAGE at the forearm skin measured by Western blotting in the troglitazone- and placebo-treated T2D patients. No changes were observed in the RAGE expression in both troglitazone- and placebo-treated groups at the exit visit (gray column) compared with baseline (black column).

Full figure and legend (36K)

Discussion

The main finding of this study was that a 12-week period of treatment with 600 mg troglitazone resulted in a considerable increase in circulating adiponectin. However, this increase had no influence on the endothelium-dependent vasodilation of the micro- or macrocirculation or on the markers of endothelial activation, such as cellular adhesion molecules. Finally, troglitazone treatment had no effect on the expression of RAGE.

The role of adiponectin in endothelial function is not well understood and is currently under intense study. An initial study indicated that, in bovine aortic endothelial cell cultures, adiponectin caused phosphorylation of the endothelial nitric oxide (NO) synthase and stimulated the production of NO (19). It is also of interest that the mechanism through which adiponectin causes increased production of NO is slightly different from the one that is related to insulin-related production of NO and vasodilation. Furthermore, adiponectin-knockout mice have impaired aortic endothelium-dependent vasodilation, suggesting a role of adiponectin in endothelial cell function (20). Finally, as mentioned in the Introduction, adiponectin has anti-atherogenic and anti-inflammatory properties; clinical studies have shown that high adiponectin levels are associated with lower risk for myocardial infarction in men (21). As a result of these observations, it has been suggested that the beneficial effects on the cardiovascular system may be related to the improvement of endothelial function (22).

In this study, we achieved a 75% increase in adiponectin levels in T2D patients after a 12-week period of treatment with troglitazone. However, this change in adiponectin levels was not associated with any improvement in endothelial function or markers of endothelial activation. Also, no association was observed between changes in adiponectin levels and changes in vascular reactivity or endothelial markers.

As we have previously described, troglitazone-treated patients with recently diagnosed diabetes had an improvement in the endothelial function of the brachial artery and a reduction in insulin levels, and these changes were strongly associated (18). In contrast, no changes were observed in measurements in patients with long-term diabetes or macrovascular disease. In this study, adiponectin levels showed a similar increase after troglitazone treatment in patients with recently diagnosed diabetes, patients with long duration diabetes, and patients with macrovascular disease. Therefore, the beneficial effects of troglitazone on endothelial function do not seem to be mediated by changes in adiponectin levels.

It is possible that the short duration of the study may be responsible for the observed results, because a study with longer duration may have shown a beneficial effect of adiponectin on endothelial function. However, we believe that this is unlikely because recent studies in our unit have shown that treatment with statins or angiotensin-converting enzyme inhibitors over the same period may induce an improvement in endothelium-dependent vasodilation (23, 24). In addition, rosiglitazone treatment over a slightly longer duration of 16 weeks also improved endothelial function in T2D patients, further suggesting that the study duration was sufficient to draw conclusions from the observed results (25). In this study, the placebo group had a higher improvement in FMD, although non-statistically significant, compared with the active group. The improvement in the placebo group is probably related to the well-known placebo effect. We believe that water retention caused by troglitazone may have minimized a comparable improvement in the troglitazone group.

The association between endothelial function and adiponectin levels has also been examined in cross-sectional studies. Initial studies indicated an association between endothelium-dependent vasodilation and adiponectin levels in healthy subjects, hypertensive subjects, and diabetic patients (20, 26, 27). However, a subsequent study failed to show any association between endothelium-dependent vasodilation and adiponectin, whereas it reported a significant correlation between endothelium-independent vasodilation and adiponectin levels (28). In this study, we failed to find any association between the baseline adiponectin levels and any of the vascular reactivity measurements at both the micro- and macrocirculation. Because the reasons for the observed results in the above studies are not well understood, more work will be required in this field before firm conclusions can be made.

Troglitazone is known to stimulate adipogenesis, resulting in weight gain and an increase in total cholesterol, LDL, and HDL (17). Pioglitazone has also been found to significantly reduce hepatic fat content and improve hepatic insulin sensitivity (16). Similar results regarding lipid levels were also observed in this study. It is also of interest that the changes in glycemia and lipid levels were the only ones that were associated with the changes in the adiponectin levels. Another interesting finding was the association between ALT, aspartate aminotransferase, and adiponectin in the baseline measurements. All of these findings, although very preliminary, suggest a role of adiponectin in liver function and are in agreement with recently published observations (16, 29, 30, 31). However, these findings are not conclusive, and more data will be required for confirmation.

Troglitazone has been withdrawn and is not being used in clinical practice anymore, and it can be argued that the observed results are not clinically important. However, we believe that the main finding of this study is that a significant rise in adiponectin levels did not result in an improvement of endothelial function. In addition, the currently available thiazolidinediones have also been shown to provide a similar increase in adiponectin levels (15, 16, 17). Therefore, we believe that the results of this study are related to the function of all thiazolidinediones and are still relevant.

No differences were found in RAGE levels, measured by Western blotting in the forearm skin biopsies, after treatment with troglitazone. Previous studies in our unit have shown a weak association between RAGE and glycemic control, but in this study, there was no change in the RAGE levels, despite a considerable improvement in the glycemia of the troglitazone-treated patients (32). Because RAGE expression is related to the production of AGEs, a slow process that requires considerable time before changes are observed, the short duration of the study may have influenced the observed results. It is also of interest that an association existed between the baseline RAGE and PPAR levels, whereas no association was found between adiponectin and RAGE levels. To the best of our knowledge, this is the first study to report such results, and further experiments will be required to fully explore this field.

In summary, we have shown that treatment with troglitazone for a 12-week period in T2D patients results in a considerable increase in adiponectin levels. However, this increase has no effect on endothelium-dependent vasodilation, indicating that adiponectin is not a major determinant of endothelial function. In addition, RAGE expression in the skin microcirculation is not affected by troglitazone treatment.

Notes

¹ Nonstandard abbreviations: T2D, type 2 diabetes; AGE, advanced glycation end-product; RAGE, receptor for advanced glycation end-product; VCAM, vascular cell adhesion molecule; PPAR, peroxisome proliferator activator receptor; FMD, flow-mediated dilation; NID, nitroglycerin-induced dilation; ALT, alanine aminotransferase; HBA_1c, hemoglobin A_1c; LDL, low-density lipoprotein; HDL, high-density lipoprotein; ICAM, intercellular adhesion molecule; NO, nitric oxide.

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Acknowledgments

This study was funded, in part, by an investigator-initiated research protocol, an American Diabetes Association research award to J. Buras, a clinical research grant to A. Veves from Parke Davis, and NIH Grant RR 01032 to the Beth Israel Deaconess Medical Center General Clinical Research Center.