Pioglitazone Increases Serum DPP-4 Level in Type 2 Diabetes Mellitus

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Introduction
Pioglitazone is a peroxisome proliferator-activated receptor-γ (PPAR-γ) agonist, and is widely used for treatment of patients with type 2 diabetes mellitus. Pioglitazone is known to increase insulin sensitivity by activating PPAR-γ in adipose tissue and to improve glycemic control [1]. Pioglitazone is considered to be unique in its ability to reduce a progression of atherosclerosis and to prevent an occurrence of cardiovascular events partly through increasing the action of adiponectin, which is one of the adipocytokines specifically and highly expressed in visceral and subcutaneous fat depots, by inducing the differentiation of adipocytes [2][3][4][5].
DipeptidylPeptidase-4 (DPP-4) is a ubiquitously expressed transmembrane glycoprotein that cleaves N-terminal dipeptides from a variety of substrates including incretin such as glucagon-like peptide-1 and gastric inhibitory polypeptide, which are released from the intestinal mucosa [6,7]. DPP-4 has gained considerable interest as a therapeutic target, and DPP-4 inhibitors that prolong the insulinotropic effect of incretin are now available for treatment of type 2 diabetes in clinical settings. Recently, it is reported that DPP-4 is expressed in adipocytes during their differentiation along with adiponectin expression and may impair insulin sensitivity directly in fat, skeletal, and smooth muscle cells [8]. In vitro experiments demonstrated that DPP-4 concentration tended to be increased by the treatment with PPAR-γ agonists [8]. These findings suggest that serum DPP-4 levels may be affected by modulating adipocyte differentiation. However, it is unknown whether or not pioglitazone affects serum levels of DPP-4 in human. Therefore, we conducted a clinical study to examine whether or not serum DPP-4 levels are affected by PPAR-γ agonist in type 2 diabetes.

Subjects
This is an observational study with 22 patients treated with pioglitazone and 15 patients with metformin, who visited Shimane University Hospital for treatments of type 2 diabetes. The patients were enrolled if informed consent was obtained after a detailed explanation of the study purpose and methods. None of them had hepatic or renal dysfunction, and taken thiazolidinedione, biguanides, or DPP-4 inhibitors so far. Pioglitazone (15-30 mg) was orally administered once daily and metformin (250mg) was two or three times after meal (500-750 mg/day) throughout 12 months. The numbers of patients who had been taking insulin, sulfonylurea, and alpha-glucosidase inhibitors were 12, 6, and 3 in the pioglitazone group, and 9, 4, and 1 in the metformin group. All prescription medications of each patient were not changed during this study. This study was approved by the ethical review board of Shimane University Faculty of Medicine, and complied with the Helsinki Declaration.
Serum DPP-4 concentration was measured by using ELISA kits purchased from R&D Systems (Minneapolis, MN). The assays were performed in duplicates according to the manufacturer's instructions. In brief, 96 wells of a polystyrene microplate were coated with anti-DPP-4 monoclonal antibody. Fifty µl of serum samples was placed in each of the 96 wells. After 2-hour incubation at room temperature, the polyclonal antibody against DPP-4 conjugated with horseradish peroxidase was used as the detecting antibody. Contents of wells were incubated for further 2 hours with substrate solution. After the reaction was stopped, the absorbance was measured at 450 nm within 30 minutes. The coefficient of variation of measurements of DPP-4 was <10.0%.

Statistical analysis
Data were expressed as mean ± SD. Wilcoxon tests were used to evaluate the effect of pioglitazone and metformin on body weight, waist circumference, HbA1c, and DPP-4 levels as compared with the data obtained at baseline. Simple correlation and multiple regression analyses were used for the relationships between two parameters. All analyses were carried out using the statistical computer program Stat View (Abacus Concepts, Berkeley, CA). P<0.05 was considered to be significant.

Baseline characteristics of patients and comparison of parameters between the pioglitazone and the metformin groups
A total of 37 patients were enrolled, with none of them withdrawing from the study. We compared various variables between the pioglitazone and the metformin groups (Table 1). No significant differences in all variables were found between them.

Chronological changes in serum DPP-4 levels, body mass index, waist circumference, and HbA1c
Chronological changes in serum DPP-4 levels, Body Mass Index (BMI), waist circumference, and HbA1c were shown in Table 2. In the pioglitazone group, BMI, and waist circumference were significantly and consecutively increased at 3 and 12 months after treatment with pioglitazone (p<0.05), while HbA 1c was significantly decreased at 3 and 12 months (p<0.01). Serum DPP-4 levels significantly increased from baseline with mean changes of 53 ng/mL [6.8% increase, 95% confidence interval (CI) 2 to 104, p<0.05] at 3 months and of 74 ng/mL (9.4% increase, 95%CI 21 to 126, p<0.01) at 12 months after treatment with pioglitazone. On the other hand, BMI, waist circumference, and serum DPP-4 levels were not changed after treatment with metformin, while HbA1c was significantly decreased at 3 and 12 months (p<0.05). Changes in serum DPP-4 level at 12 months were significantly greater in the pioglitazone group than in the metformin group (Figure 1).

Correlations between percent change in serum DPP-4 concentration versus baseline and changes in values of each parameter
We performed correlation analysis between percent (%) change in serum DPP-4 concentration versus baseline and % change in values of each parameter at 12 months in order to investigate which parameters could be associated with serum DPP-4 levels (     DPP-4 was significantly and positively correlated with % change in BMI ( Figure 2), but not other parameters. Then, multiple regression analysis adjusted for age, duration of diabetes, serum creatinine, and HbA1c showed that the positive association between % change in DPP-4 and % change in BMI was still significant (β=0.59, p=0.013).

Discussion
DPP-4 is considered as a negative regulator of glucose tolerance by inactivating incretin action. In addition, blood levels of DPP-4 were elevated in poorly controlled diabetes [10]. To our knowledge, there are no clinical studies investigating the effect of PPAR-γ agonists on serum DPP-4 levels. In this study, we found for the first time that pioglitazone increased serum DPP-4 levels in type 2 diabetes.
A recent study showed that DPP-4 is expressed in adipocytes and induces insulin resistance directly, and that serum DPP-4 levels were associated with visceral fat accumulation and metabolic syndrome [8]. These data suggest that DPP-4 is one of the adipocytokines and that serum DPP-4 levels may be affected by adipogenesis. Since PPAR-γ is a master regulator of adipogenesis and its activation induces mesenchymal stem cells into adipocytes, PPAR-γ agonists might affect DPP-4 expression and secretion in adipocytes. In this study, serum DPP-4 levels were slightly but significantly increased after treatment with pioglitazone, while they were not changed in patients treated with metformin. Moreover, changes in serum DPP-4 levels were associated with changes in BMI. These findings suggest that increased serum DPP-4 by pioglitazone might be derived from adipose tissue.
The relationships of serum DPP-4 increased by pioglitazone with glucose metabolism and diabetic complications is unknown. Previous studies indicated that treatment with pioglitazone decreased the incidence of cardiovascular disease [2]. The favorable effects of pioglitazone on cardiovascular disease might be its pleiotropic effect. On the other hand, pioglitazone is reported to increase the risks of heart failure and osteoporotic fracture [11,12]. Several studies showed that inhibitions of DPP-4 improved heart failure after cardiovascular infarction as well as osteoporosis fractures [13,14]. Although the mechanism of the increased risks of heart failure and osteoporotic fracture by pioglitazone is still discussed, these findings suggest that increased DPP-4 levels by pioglitazone might be involved in the pioglitazone-related adverse events. A previous animal study showed that DPP-4 inhibitors suppressed pioglitazone-induced gain of body weight, suggesting that combined therapy of pioglitazone with DPP-4 inhibitors might be useful to lead the beneficial effects of pioglitazone without adverse effects [15].
In conclusion, this is the first report showing that pioglitazone treatment increased serum levels of DPP-4 in patients with type 2 diabetes. However, this is a small observational study to investigate the effect of pioglitazone on serum DPP-4 levels. We thus need further large scale clinical trials to confirm our findings.