br Materials and methods br

Materials and methods
Results
Discussion In this study, we confirmed the activation of Akt by udenafil in 3T3-L1 cells. To elucidate the underlying mechanism of udenafil, we focused on the mitochondria. Our findings demonstrated that udenafil improved mitochondrial function and expression of genes involved in mitochondrial biogenesis and beta-oxidation in 3T3-L1 cells. This might be the mechanism underlying PDE5 inhibitor-enhanced insulin signaling in adipocytes. Adipocytes play a key role in maintaining glucose homeostasis, and abnormal cells are known to cause multiple metabolic problems including diabetes [20]. Cell experiments have shown an increase in the number of mitochondria in 3T3-L1-differenciated white adipocytes [21], [22]. The need for elevated mitochondria levels in white adipocyte differentiation implies that mitochondria are essential for the functioning of adipocytes. The proportional relationship between adipocyte function and mitochondrial production is consistent with the hypothesis that adipocyte dysfunction in type 2 diabetes is associated with defects in mitochondria. When mitochondrial activity is reduced, fatty RepSox oxidizing ability is lowered, and levels of acyl-CoA and diacylglycerol, which are not oxidized, increase in the cells. These molecules activate protein kinase-C and induce phosphorylation at the Ser307 site of insulin receptor substrate-1 (IRS-1). Due to this phosphorylation, IRS-1 fails to bind the insulin receptor and cannot facilitate the activation of phosphatidylinositol 3-kinase (PI3K), resulting in the inhibition of glucose uptake. Eventually, elevated level of intracellular fatty acids due to defects in the mitochondria can interfere with insulin signaling. Accordingly, studies have been conducted to show the importance of adipose tissues for glucose uptake. In studies using db/db mice, reductions in the number of mitochondria, mitochondrial respiration, and FAO were observed in adipose tissues [23]. Mitochondrial function has also been founded to decrease in ob/ob mice and adipocytes of diabetic patients [24], [25]. The present study demonstrated that udenafil increased expression of the mitochondrial OxPhos gene. In particular, the increase in complex I proteins was substantial. Dysfunction in complex I, the first enzyme of the respiratory chain, can increase oxidative damage. Furthermore, increased reactive oxygen species (ROS) has been associated with some mitochondrial diseases [26]. In a study using diabetic mice, treatment with tadalafil decreased ROS production through complex I [27]. Thus the increased mitochondrial function shown in this study may be associated with an increase in complex I activity. Furthermore, in our study, udenafil treatment increased the expression of PGC-1α, a key regulator of mitochondrial biogenesis. PGC-1 is known to regulate mitochondrial production and has a role in decreasing insulin resistant states in human adipose tissues [28]. PGC-1 functions by binding to transcription factors to increase their activity. More specifically, PGC-1α can bind to PPAR-α and PPAR-β to increase the expression of genes involved in FAO. It also binds to the nuclear respiratory factor 1 (NRF-1) to upregulate the transcription of OxPhos-related genes and promote mitochondrial proliferation and DNA transcription, thereby increasing mitochondrial production [29]. The association of PDE5 inhibitors with insulin resistance and diabetes has been continuously discussed, although the literature on the mechanism of action is limited. Therefore, we investigated changes in mitochondrial and function after udenafil treatment in order to provide insight into the mechanisms mediating this relationship. We found that udenafil significantly increased OxPhos gene expression and mitochondrial OCR, indicating an enhancement in mitochondrial function. We also demonstrated that udenafil can stimulate key regulators of FAO in 3T3-L1 cells.
Conclusion