The Roles Of Tetrahydrobiopterin In Regulating Glucose Metabolism In Endothelial Cells And Promoting Wound Healing In Diabetic Mice

Tetrahydrobiopterin (BH4) is an essential cofactor required for theactivity of endothelial nitric oxide synthease (eNOS) in endothelial cells,which plays a role in increasing NO production, decreasing SO production andthen restoring the diabetic endothelial dysfunction. BH4has been regarded asa rational therapeutic target in diabetic vascular disease states. However, thefurther mechanism still remains unclear.Our previous study shows that BH4synthesis promotes angiogenesis,which depends on a PI3K/Akt-positive feedback loop through NO production.The PI3K/Akt pathway is considered to be the major effector ofglucose-metabolic insulin action, which provides a possible mechanism ofregulating glucose metabolism for BH4in endothelial cells. Angiogenesis is akey phase of wound healing, which shows that BH4may promote the woundhealing in diabetic mice.In this research, we explore the possibility and signaling mechanism ofBH4regulating glucose metabolism in endothelial cells. First, in low glucose(5.5mM) environment, we found that sepiapterin (Sep)-based BH4synthesisup-regulates glucose metabolism. Based on data of the lactate concentrationand lactate metabolism-related proteins, BH4synthesis may increase TCAprocess in BAEC. In HUVEC and BAEC, we knew that BH4synthesis bothcan activate time-dependently Akt and Erk1/2signaling pathways. The next,the BAEC monolayer were incubated for30min with LY294002and PD98059,the inhibitors of PI3K/Akt and Erk1/2. The result shown that the role of BH4in regulating glucose metabolism, at least in part, depends on PI3K/Aktpathway; and the Erk1/2pathway may crosstalk with the PI3K/Akt, due to theinhibition of p-Erk1/2and a concomitant increase in p-Akt. Finally, BAECtreated with SSZ (inhibitor of sepiapterin reductase) and L-NAME (an analog ofL-arginine, eNOS inhibitor) for48h. The result shown that BH4synthesis up-regulates Akt pathway and glucose metabolism via NO in low glucoseenvironment; but in high glucose (30mM), Sep up-regulates Akt pathway andglucose metabolism via NO-independent process. In hypoxia (1%O2)environment, Sep could still increase Akt-glucose metabolism.Altogether, we revealed that Sep-based BH4synthesis up-regulatesglucose metabolism, which depends on PI3K/Akt pathway via NO production;but in HG, which may be NO-independent.In animal test, we explore the promoting efficacy of BH4in diabeticwound healing. Our data showed that when promotion of wound closure wascompared on the same day, the Sep-treated group versus DMSO-treated grouphad stronger effect of wound healing at day6and day9in normal mice and atday9in diabetic mice.