The major findings of our present study are: (1) an antidiabetic treatment with insulin or metformin inhibits the development of hypoadiponectinemia and downregulation of APPL1 in ZDF rats, but (2) both treatments are not able to improve adiponectin-induced vasodilation and endothelial dysfunction; (3) treatment with insulin is able to control blood glucose levels in ZDF rats, but (4) causes a decrease of AdipoR1, AdipoR2 and eNOS expression in small mesenteric resistance arteries; (5) treatment with metformin even in highest tolerable doses does not decrease blood glucose levels in ZDF rats and (6) reduces expression of AdipoR2 in mesenteric arteries.
Effect of antidiabetic treatment on adiponectin-induced vasodilation and endothelial function
In our recent study  we found that adiponectin provokes a NO-dependent vasodilation of mesenterial resistance arteries in ZL rats, which is diminished in arteries of ZDF rats. The expression analysis of different components of the adiponectin signaling pathway showed a significant downregulation of APPL1 in mesenteric arteries of ZDF rats. In addition ZDF rats exhibited hypoadiponectinemia and endothelial dysfunction. Therefore, we concluded that hypoadiponectinemia itself and a reduced intracellular adiponectin signaling are partly responsible for endothelial dysfunction in diabetes mellitus type 2. This hypothesis was supported by a study of Ouchi et al. , who found that adiponectin-knockout mice exhibit endothelial dysfunction and another study of Cao et al. , in which a replenishment of adiponectin in knock-out mice normalized endothelial function. Similar Deng et al.  could show that endothelial dysfunction in high-fat diet fed rats was ameliorated by an in vitro incubation of aortic rings with globular adiponectin in high doses. Therefore interventions directed to increase adiponectin levels in diabetes mellitus type 2 are possibly able to improve endothelial function. Against our expectations in the present study an antidiabetic treatment with insulin or metformin could not improve the vasodilatory effect of adiponectin and endothelial function in diabetic ZDF rats (Figure 2+4), although both restored adiponectin levels and also increased the expression of APPL1 in ZDF rats to levels of nondiabetic ZL rats (Figure 1+6). Thereby, it has to be mentioned that these results of course only apply to our ex vivo setting and that we can`t make a statement to possible in vivo effects of the tested antidiabetic treatment.
In insulin-treated animals the reason is probably the observed downregulation of both adiponectin receptors (Figure 5) and eNOS (Figure 7), which with their localization at the beginning and end of the adiponectin signaling pathway are the main regulators of adiponectin sensitivity and NO-production. Therefore, the reduced expression of the adiponectin receptors and eNOS might diminish the benefical effects of increased adiponectin serum and APPL1 expression levels. Additionally, the increase of APPL1 expression might be counteracted by the complementary increase of APPL2 expression, even if not statistically significant. The fact that an early treatment with insulin in diabetes mellitus type 2 does not improve endothelial function is in line with a recent study in human, in which flow-mediated dilation remained unchanged after initiation of insulin therapy .
For metformin-treated animals our study suggests two possible reasons for the absent improvement of vasodilation. On the one hand the reduced expression of AdipoR2 (Figure 5b) and on the other hand the uncontrolled blood glucose levels. Thereby, decreased expression of AdipoR2 may at least in part reduce adiponectin sensitivity resulting in decreased eNOS activation and NO production. The fact that metformin is not able to control blood glucose in ZDF rats was already observed in another study, since ZDF rats develop insulin deficiency between an age of 14 and 21 weeks . An uncontrolled diabetes mellitus may cause endothelial dysfunction through additional mechanisms apart from adiponectin signaling. Accordingly, a study in another rat model of diabetes mellitus type 2 showed an improvement of endothelial function in aortic rings after metformin therapy, but this effect was associated with a significant improvement of blood glucose levels . However, in another experiment metformin therapy improved endothelial function independent of a blood glucose lowering effect, but this study was performed in streptozotocin-induced diabetic rats, a model of diabetes mellitus type 1 .
Effect of antidiabetic treatment on adiponectin levels and expression of components of the adiponectin signaling pathway
As mentioned above, both antidiabetic treatments inhibited the development of hypoadiponectinemia during the animal experiment (Figure 1).
Generally, the effect of insulin on adiponectin serum levels is contentious. In vitro, insulin inhibits the expression of adiponectin at mRNA level , but then it leads to an increased adiponectin secretion from adipocytes [36, 37]. In vivo, there is a good documented association of hypoadiponectinemia with insulin resistance and diabetes mellitus type 2 , but the effect of insulin therapy on serum levels of adiponectin is not well studied so far. A study in five healthy male volunteers showed a decrease of adiponectin levels during a hyperinsulinemic euglycemic clamp . Another study with insulin treatment in a murine model of obesity and diabetes mellitus type 2, the db/db mice, suggested no impact on adiponectin levels . A recent study indicates an elevation of adiponectin levels in a hyperinsulinemia rat model . In this respect, our study supports new evidence for a positive effect of insulin therapy on adiponectin levels despite of an increase in body weight.
For the effect of metformin on adiponectin levels there are more studies available. In vitro metformin increased the expression and secretion of adiponectin in human adipose tissue samples . However, results of studies in humans with metformin treatment for different diseases showed mostly no effect on adiponectin serum levels [43–48], but it also exists evidence for an adiponectin-raising effect [49–51]. In this context our animal study supports a benefical effect of metformin on adiponectin levels.
Considering the adiponectin receptors, we found under insulin treatment a significantly decreased expression of AdipoR1 and AdipoR2 compared to untreated animals (Figure 5). On this so far data from an in vitro study exist showing a reduced expression of both adiponectin receptors in skeletal and vascular smooth muscle cells by insulin . Another recent study also revealed that an insulin infusion in a rat model decreased expression of AdipoR1 in skeletal and myocardial muscle . This effect seems to be mediated by a direct repressive effect of insulin on the promoter activity of AdipoR1 . Our data match these studies and further support and expand these findings to the vasculature. The effect of metformin on adiponectin receptor expression was so far studied in adipose tissue, skeletal muscle and liver of ZDF rats . In this experiment a significant increase for both receptors in skeletal muscle was detected and our study now expands these findings with expression data for small mesenteric resistance arteries showing a decreased expression of AdipoR2.
To our knowledge, with the present study we provide first data on the effect of an antidiabetic treatment on the expression of APPL1 and APPL2 showing that insulin as well as metformin is able to increase the expression of APPL1 in resistance arteries of ZDF rats, while APPL2 expression remained unchanged (Figure 6).
In respect to eNOS, we found that insulin treatment significantly reduced its expression in mesenteric arteries (Figure 7). This goes with findings of Zanetti et al., who showed that an overexpression of eNOS in diabetic animal model can be attenuated by insulin treatment . However, another study in ZDF rats found no effect of an insulin therapy on eNOS expression in the aorta . Metformin treatment did not alter eNOS expression in our experiment, which is in line with studies in other animal models of diabetes mellitus .
However, our expression analyses are performed with real-time RT-PCR and therefore only reflect expression at mRNA level, since second order mesenteric arteries of rats do not yield enough protein for a reliable western blot analysis.