PAHSAs has been reported to reduce blood glucose levels, enhance insulin sensitivity, and anti-inflammation in different diseases[3, 7, 8, 26, 27]. However, there was contradictory evidence showing that 5-PAHSA or 9-PAHSA isomers did not improve glucose control in mice. These contrary studies may in large part due to the methodology chosen, but different PAHSAs isomers likely play a part as well. Therefore, we developed a new synthesis pathway separating the enantiomers of 9-PAHSA into S-9-PAHSA and R-9-PAHSA, and investigated their biological activities in SH-SY 5Y cells, and effect of S-9-PAHSA on glycolipid metabolisms in T2DM mice.
In this study, we found S-9-PAHSA could improve insulin resistance in HFD-induced T2DM mice, but it had no significant effect on fasting blood glucose, glycosylated serum protein and weight. Furthermore, the administration of S-9-PAHSA had no effect on release of GLP-1, an important hormone that stimulates insulin secretion[29, 30]. This implied that the anti-diabetic effect of S-9-PAHSA might not be exerted by reduction in glucose level, weight or directly GLP-1 secretion, while improvement in insulin resistance could be one possible reason for this phenomenon.
T2DM is mainly manifested insulin resistance. There is increasing evidence that insulin resistance is strongly induced by autophagy impairment. It was recently demonstrated that the role of insulin resistance and autophagy in some neurodegenerative diseases is crucial, and decreased autophagy could increase the risk of insulin resistance during the skeletal muscle aging process. Impairment of autophagic flux in muscle cells induced insulin resistance directly. Fujitani et al. reported that reduced insulin secretion was closely related with the level of autophagy. In our studies, the effects of S-9-PAHSA on enhancing autophagy were revealed in SH-SY 5Y cells, and improvement of insulin resistance via elevating the activity on autophagy might be an important aspect of S-9-PAHSA’s metabolic benefits, which is consistent with a previous study of PAHSA on hepatic and systemic insulin sensitivity.
Interestingly, we also found that the treatment of S-9-PAHSA could significantly inhibit the abnormal ox-LDL in the serum of HFD-induced T2DM mice, suggesting that S-9-PAHSA might have a promising effect on lipid metabolism disorders. Similarly, in our previous research, 5-PAHSA also exerted positive influence on lipid metabolism in DB/DB mice. Many studies have verified that the stimulation of ox-LDL was related to suppression of autophagy. The enhancement autophagy could effectively reduce ox-LDL-induced RAW264.7 foam cell formation, reducing cellular lipid accumulation and delaying cell senescence. Studies showed high glucose stimulated LDL transcytosis by a novel CAV1-CAVIN1-LC3B signaling-mediated autophagic degradation pathway. Since T2DM patients usually suffered glucose metabolism disorders, low-density lipoprotein (LDL) could be saccharified by sustained hyperglycemia, then being swallowed by monocytes or macrophages, and causing intracellular cholesterol accumulation, and formatting foam cells that accelerate the development of atherosclerosis[38, 39]. At the same time, insulin resistance could cause abnormal activation of the lipid synthesis pathway, which in turn resulted in lipid deposition. In addition, there is intense oxidative stress in lipid metabolism disorders. Recent studies have identified that the human umbilical vein endothelial cells exposed to ox-LDL exhibited enlarged ROS production. Our study results demonstrated that the treatment of S-9-PAHSA could significantly decrease the level of ROS, suggesting that S-9-PAHSA might play a protective role through anti-oxidative stress. Thus, targeting the crossed pathway between enhancement of autophagy and inhibition of ROS production may be a promising strategy with respect to the lipid metabolism after treatment of PAHSA.
Currently, autophagy, a metabolic pathway, has deserved widespread attention. It is beneficial to maintain the dynamic balance of cells. There are a great deal of triggering factors, including ROS, LDL, inflammatory factors, etc, among which could cause the changes of autophagy regulation. In our previous research, especially, it was confirmed that autophagy was involved in the development of T2DM-associated cerebrovascular disease and cognitive dysfunction[44, 45] and 9-PAHSA could promote autophagic flux in diabetic mice. Previous reports demonstrated the suppression of high glucose on the activity of AMPK, which then consolidated BCL2-Beclin1, leading to autophagy inhibition in cardiomyocytes. LC3II/I and Bcl-2, could signify the autophagic level. Specifically, when defective autophagy occurs, the expression of LC3II/I and Bcl-2 is down-regulated. In our experiments, through the stimulation of high glucose and lipid, the apparent decrease of LC3II/I and Bcl-2 were found, indicating that the autophagy level was decreased under the diabetic environment. However, this trend was reversed by the supplement of S-9-PAHSA, indicating that S-9-PAHSA might be helpful in preventing the glucose and lipids metabolism disorder via the alternation of autophagy.
Caspase family and Bcl-2 family proteins are key regulators in apoptosis[47, 48]. BAX and Bcl-2 are the major factors in the regulation of the mitochondria-mediated pathway of apoptosis. The ratio of BAX/Bcl-2 is a critical determinant of susceptibility to apoptosis, rather than the levels of individual proteins. S-9-PAHSA might have an effect on apoptosis via caspase family, rather than Bcl-2 family, which was supported by our results that the levels of Cleaved caspase-3 were significantly decreased after S-9-PAHSA administration, but the ratio of BAX/Bcl-2 in GF + S-9-PAHSA group did not change compared with GF group.
This study showed that S-9-PAHSA increased protein expressions of PI3K and phosphorylation of AKT in SH-SY 5Y cells exposed diabetic environment, and this compound increased the level of autophagy, as well as reversed suppression on oxidative stress. The PI3K/AKT signaling pathway is a major mediator of insulin effects and plays a crucial role in T2DM pathogenesis. In patients with T2DM, alterations in the PI3K/AKT pathway primarily manifested as decreased phosphorylation. Glucose transporter type 4 (GLUT4), mediated by the PI3K and AKT signaling pathways, also plays an important role in maintaining blood glucose homeostasis. Numerous studies found that the level of autophagy was enhanced by the alternation of PI3K/AKT signal way[52, 53]. These results hinted that PI3K/Akt pathway was responsible for the protective actions of S-9-PAHSA in diabetic glycolipid metabolism dysfunction. However, further experiments are needed.