Mechanism Of Liver Metabolism Regulated By Hippo Pathway And Autophagy In The Newborn Piglets With Intrauterine Growth Restriction

Intrauterine growth retardation (IUGR) is defined as impaired growth and development of the mammalian embryo/fetus or its organs during pregnancy, remains a major problem for both human health and animal production because of its association with high rates of neonatal mortality, low efficiency of food utilization, permanent adverse effects on postnatal growth and development. Currently, the mechanism of IUGR is poorly understood, which leads to the insufficient treatment and prevention of IUGR. Thus, to fully understand the occurrence and regulation of IUGR is important. Hippo signaling pathway is a novel pathway which plays an important role in the mammals’ organ size control, cell growth and development. In addition, autophagy is well known for the physiological significance in the stress conditions and early stage of animal development. The purpose of this study is to investigate whether the Hippo signaling pathway and autophagy are involved in the liver metabolism regulation of IUGR in newborn piglets, primarily reveal its mechanism, and provide a theoretical basis for the understanding of IUGR.In this study, the impact of IUGR in the liver metabolism of newborn piglets was analyzed. The activities of Hippo, autophagy and their related pathways in the livers of newborn piglets were examined through molecular biology and cell biology experimental techniques. The main results are as follows:(1) The liver/body weight ratio of IUGR newborn piglets was significantly decreased compared to the normal newborn piglets (P<0.05), indicating that while IUGR occurs, the liver growth is arrested. BCAA/AAA ratio was significantly reduced in the plasma free amino acids (P<0.05); and the IUGR livers display a disorganized arrangement of hepatocytes, many of which have an abnormal clear cytoplasm with H&E staining. Together, these results have been pointed out that the livers of IUGR newborn piglets had been damaged. The IUGR newborn piglets demonstrated that plasma glucose was significantly decreased compared to the normal newborn piglets (P<0.01), and it showed deficiency of glycongen in the IUGR livers through the lighter in color of PAS staining, in addition, we found that the IUGR liver had a notable increase in AMPK activation (P<0.01). These results suggest that IUGR can alter the enenrgy homeostasis and change glucose uptake/utilization efficiency in the newborn piglets(2) We examined the Hippo signaling pathway in the livers of IUGR newborn piglets with Western blot and immunohistochemistry. MST and LATS1activities were significantly increased in the IUGR livers (P<0.01), and also the phosphorylation levels of YAP (P<0.01), suggesting that the activity of Hippo was increased in the livers of IUGR newborn piglets.(3) Examined by Western blot and transmission electron microscopy, autophagy activity in the livers of IUGR newborn piglets was reduced. We used transmission electron microscopy to detect the number of autophagic vacuoles in the IUGR livers and found it decreased significantly (P<0.01), Western blot further demonstrated that the autophagy activity was decreased significantly in the IUGR livers (P<0.01).(4) Western blot analysis revealed that the activities of Hippo, autophagy and their related pathways—Wnt and mTOR were significantly reduced in the livers of IUGR newborn piglets (P<0.01). Interestingly, we found that in the IUGR livers, Dv12protein of Wnt signaling pathway was aggregated and the levels were significantly increased (P<0.01).(5) In order to investigate the mechanism of autophagy activity reduced in the livers of IUGR newborn piglets, we examined mTOR-AMPK-ULKl interaction with Western blot and found that the phosphorylation levels of ULK1Ser555were significantly reduced in the IUGR livers (P<0.01), and in both groups, Ser757didn’t showed any phosphorylation. It indicates that AMPK and ULK1interaction was deficient in the IUGR livers.In summary, a conclusion came up that the activities of Hippo signaling pathway is increased in the livers of IUGR newborn piglets conpared to the normal ones, suggesting that Hippo signaling pathway may be involved in the formation of IUGR. Moveover, the phosphorylation of ULK1through AMPK is deficient and then lead to the failure of autophagy induction, which may be related to the aggregation of Dv12, this process may be a critical point in IUGR formation.