Influence of insulin and glargine on outgrowth and number of circulating endothelial progenitor cells in type 2 diabetes patients: a partially double-blind, randomized, three-arm unicenter study

Endothelial progenitor cells (EPCs) are immature bone marrow-derived cells which undergo differentiation into endothelial cells and participate in endothelial repair and neoangiogenesis [1],[2]. Clinical trials have shown that the number of circulating EPC correlates with cardiovascular risk factors and is an independent predictor of cardiovascular death [3],[4]. Patients with diabetes type 1 and 2 have a reduced differentiation and function of EPC in vitro [2],[5],[6].

Poor glycemic control and increase of glycated hemog-lobin level is associated with an increase in the risk of cardiovascular events [7], death [8],[9] and for the development of retinopathy or renal failure [9]. Data from clinical trials imply that there might indeed be positive effects of insulin therapy for the microvasculature [10] while there seem to be limited effects on macrovascular events in the ORIGIN and UKPDS trials [11],[12]. Insulin has previously been shown to have positive effects on micro- and macrovascular function in experimental clinical trials [13]-[16]. So far it is still not known, whether the improvement of glucose control by any available therapeutic strategy would have the same effects.

We were previously able to show that intensified insulin therapy in poorly controlled diabetes leads to increased numbers of CD34/CD133-positive progenitor cells that can also be considered as precursors of EPC [17]. In addition, we could show that insulin stimulates the in vitro outgrowth of EPC and tube formation of adult endothelial cells in an IGF-1 receptor dependent manner [18]. Hence, we hypothesized that insulin could improve the outgrowth and number of circulating EPC independent of glucose levels and thereby improve micro- or macrovascular function.

In this controlled trial it was therefore studied, whether early addition of the long acting insulin analogue glargine or NPH-insulin are capable of increasing the number and differentiation of EPC compared to an escalation of oral diabetes medication after 4 months of treatment. In addition, the effects of insulin on microvascular function as well as carotid intima media thickness were studied in this randomized and controlled setting.

10 out of 73 randomized patients stopped the trial before the first visit and therefore no values for EPC were obtained. 3 patients discontinued before visit 2, 4 patients had missing values for EPC after 4 weeks and one patient had a value of 0.00 and methodological bias at visit 1. Therefore the intention to treat population included 55 patients; 13 patients in the group with oral medication; 20 patients receiving insulin glargine and 22 patients receiving NPH Insulin (Figure 1).

No significant differences between the groups concerning other metabolic parameters such as triglycerides, LDL and HDL cholesterol, body mass index or blood pressure could be observed. The number of reported adverse events including hypoglycemia, and serious adverse events did not differ between the groups.

After four months of treatment, a mean relative change of 2.6 (+/-6.8) was obtained in the intention to treat analysis set with median time between visit 1 and 3 of 119 days. The mean relative change was 1.3 (+/-3.0) in the glargine group, 3.1 (+/-7.4) in the NPH insulin group and 3.8 (+/-9.5) in the oral group. No statistically significant differences between the three groups could be shown (p = 0.55, Table 3).

The number of colony forming units (CFUs) of EPC as detected by in vitro outgrowth were significantly higher in the NPH insulin group (29.2+/-6.4) and the glargine group (29.4+/-6.7) compared to the oral group (23.2+/-6.3) after 4 months (p = 0.013, Figure 2a). When data from all patients receiving insulin (NPH or glargine) were compared to those on oral medication a significant effect could be shown (p = 0.003, Figure 2b).

Mean intima media thickness (IMT) at visit 1 was 0.80 mm (+/-0.14) with mean IMT of 0.79 mm (+/-0.13), 0.79 mm (+/-0.17) and 0.83 mm (+/-0.13) in the glargine, NPH insulin and control group, respectively (p = 0.737, Table 1). At visit 3 mean IMT decreased to 0.76 mm (+/-0.12) with mean IMT of 0.74 mm (+/-0.10), 0.76 mm (+/-0.14) and 0.82 mm (+/-0.11) in the glargine, NPH insulin and control group, respectively (p = 0.153, Table 2) and therefore no statistical significance was shown. However, when after 4 months IMT data from all patients receiving insulin (NPH or glargine) were compared to those on oral medication a trend towards a reduction of IMT by insulin treatment could be documented (p = 0.06) indicating that in a longer trial or a larger study population a significant result might have been achieved (Figure 2c). Furthermore the decrease of IMT from 0.80 mm (+/-0.14) at visit 1 to 0.76 mm (+/-0.12) at visit 3 in all patients was statistically significant (p = 0.03) indicating that all patients profited from the intervention with respect to IMT.

Concerning the microvascular function of the skin at visit 1 a mean of 462.0% (+/-205.5) increase upon heat provocation could be observed, with a mean increase of 408.4% (+/-216.5), 455.7% (+/-171.8) and 555.1% (+/-223.5) in the glargine, NPH insulin and control group, respectively (Table 1). At visit 3 a mean of 515.7% (+/-283.8) increase upon heat provocation could be seen with a mean increase of 514.5% (+/-329.7), 487.7% (+/-229.6) and 564.9% (+/-306.5) in the glargine, NPH insulin and control group, respectively (Table 2). No significant difference between the three groups could be observed.

18 adverse events were reported. 5 in the glargine group, 7 in the NPH insulin group and 6 in the control group. Of those there were 2 mild hypoglyceamias reported in the glargine group. No adverse effects was categorized as severe.

This is the first study investigating the effects of add-on basal insulin treatment in a direct comparison to oral diabetes medication on endothelial progenitor cells in patients with type 2 diabetes mellitus. Our current data showed that a 4-month treatment with insulin glargine or NPH insulin significantly increases the ex vivo differentiation of EPCs in patients with type 2 diabetes mellitus, compared to an escalation of the oral antidiabetic therapy without additional insulin treatment. These findings are in agreement with our previously published data showing that insulin glargine and human insulin stimulate the clonogenic potential of EPCs in vitro [18]. However, no significant change in the numbers of circulating EPCs could be observed within the 4 months suggesting that there are no direct biological effects of insulin on the number of circulating EPC at least within the documented 4 months treatment in this study. These results are in agreement with a study by Fadini et al. where significant changes after adding insulin analogues in a crossover design could only be shown after 6 months of treatment [28]. As we previously described in a pilot study [17], a very recent study has shown an increase on circulating EPCs and outgrowth of CFUs in patients with long-standing uncontrolled type 2 diabetes receiving an intensified antidiabetic treatment. Yet this study did not compare different treatment strategies [29], which was the aim of our current study. Our data indicate that insulin treatment has a possible early effect on the functionality of EPCs while changes in numbers of circulating EPC could require more time and a long term improvement of metabolic control.

It has been previously shown that hyperglycemia reduces survival and impairs the function of EPCs in vitro [30] and that in patients with type 2 diabetes mellitus the levels of circulating EPCs are closely related to glycaemic control [31]. Poor glyceamic control is known to play a key role in the properties of circulating EPCs and several mechanisms seem to be involved in that, including advanced glycation end products formation [32] and reduced activity of silent information regulator 1 (SIRT1) and increased synthesis of platelet-activating factor (PAF) [33]. All 3 groups in this study achieved a significant decrease of HbA1c. However as aimed for, there were no significant differences concerning the HbA1c levels between the groups at baseline and after 4 months suggesting that the observed effects on EPC outgrowth did not depend on glycemic control but rather on direct insulin effects as we were previously able to show in vitro [18].

Since glargine and NPH insulin have a different structure and biologic activity, we assumed that the different pharmacokinetic properties could translate into different effects on EPCs. We were previously able to show that insulin stimulates the clonogenic potential of EPCs by IGF-1 receptor-dependent signaling [18] and it is known that insulin glargine has a 6-fold increased affinity for the IGF-1 receptor [34]. Yet the glargine metabolite gly-A21-insulin, that has been shown to have similar affinity to the IGF-1 receptors as human insulin [34], comprises most of the available substance in vivo [35]. Accordingly and in line with our previous in vitro data [18] we found no differences between NPH insulin and glargine on the function or the number of circulating EPCs. Furthermore no differences concerning other metabolic parameters as cholesterol levels, body mass index, skin microvascular function and in terms of safety profile including hypoglycemia could be observed.

IMT has been widely used as a surrogate parameter in primary intervention studies of cardiovascular risk reduction [36]. Previous studies have shown that patients with type 1 diabetes mellitus without macrovascular disease have low numbers of circulating EPCs and an increased IMT [37]. Moreover a previous study in middle-aged healthy people found a significant correlation between EPC number and IMT [38]. We were not able to detect any significant changes in carotid intima media thickness throughout the study between the 3 study groups. However, interestingly when IMT data from all patients receiving insulin (NPH or glargine) were compared to those on oral medication 4 months after baseline, a trend towards a reduction of IMT by insulin treatment could be documented indicating that in a trial with a larger study population a significant result might have been achieved. This hypothesis can be further supported by data recently published showing that in people with cardiovascular disease and/or CV risk factors, insulin glargine used to target normoglycemia modestly reduced carotid IMT progression [39].

Remarkably, at least a trend for a reduction of IMT was seen in patients treated with insulin while there were no effects of NPH insulin or glargine treatment on microvascular function. These data in our randomized and controlled setting contrasts previously published results from type 2 diabetes patients subjected to glycemic improvement by insulin or oral treatment [40]-[42]. It will have to be clarified, whether this is due to differences in study design or a consequence of methodological aspects. Yet, we believe that our data suggests that there could be only marginal effects of insulin treatment or improvement of glucose control since we did not see effects after 4 months of treatment even when patients in the study were pooled (data not shown).

Limitations of the study certainly include the relatively high rate of drop-outs which led to a reduction of the power of the study as mentioned above. Drop-out rates were relatively high mainly because patients that were randomized to receive insulin treatment would then decide to leave the study probably because they initially believed they would enter the oral medication arm although they were thoroughly instructed that this was a randomized trial. Additionally it should be noted that our current clinical study was based on a methodological paper that has been previously published [18]. Since then, different approaches have been used to culture and quantify EPC therefore the term EPCs should be used with caution since most commonly used methods for isolating EPCs in culture have been found to generate a mixed population of cells [43] However, we were previously able to show that the cells in our outgrowth assay carry established progenitor cell antigens, have a distinct influence on tube formation in vitro and incorporate into vascular structures and therefore should be considered angiogenic progenitors [18]. Even though the goal of this study was to detect possible differences on EPCs between patients on guideline-suggested diabetes oral medication or add-on therapy with insulin glargine and NPH insulin, the fact that in most cases the diabetes medication in the oral group was escalated could be a limitation since many oral antidiabetics have been known to have at least some effects on EPCs [44]. It is in most cases though not clear, whether these effects are direct drug effects or if they are a result of improved glycemic control. Furthermore several anti-diabetic and cardioprotective agents including metformin and rosuvastatin have been shown to have beneficial effects on endothelial cell viability, regeneration and apoptosis [45]. Even newer anti diabetic medication as the dipeptidyl peptidase-4 inhibitor Saxagliptin has been shown to improve the function of circulating pro-angiogenic cells from type 2 diabetic patients [46]. Glitazone treatment and a change in lipid lowering therapy, that were known at the time the study was designed to influence EPC, were not allowed in this study.

The results of this study are independent of glycemic control but nevertheless the functional meaning of an improved EPC differentiation still needs to be clarified.

The study shows that a 4-month treatment with insulin glargine or NPH insulin significantly increased the outgrowth of EPCs in patients with type 2 diabetes mellitus compared to patients who received an escalation of the oral diabetes treatment only. The observed effects on EPC outgrowth did not depend on glycemic control but rather on direct insulin effects. We therefore believe that this study provides valuable information regarding pleiotropic effects of insulin that could possibly influence vascular homeostasis and outcomes.

D.O. served as principal investigator, consulted participants in the outpatient clinic and wrote the manuscript. R.v.B. Z.D. and S.K. reviewed and edited the manuscript. S.V. performed analysis of samples, reviewed and edited manuscript. A.S. and S.E. had full access to all of the data of the study after database closure and performed statistical analyses, A.S. reviewed and edited the manuscript, R.C. and G.K. reviewed and edited the manuscript. M.M. contributed to discussion, patient recruitement and reviewed the manuscript. P.P.N reviewed and edited the manuscript. P.M.H. as principal investigator was responsible for study design, concept, analyses and writing of the manuscript. All authors thank the patients for participation in the study. All authors read and approved the final manuscript.