APJ is mainly coupled to PTX

APJ is mainly coupled to PTX-sensitive Gi/o proteins [33], and thus, it is associated with the inhibition of adenylyl cyclase and the activations of PI3K/Akt signaling and MAPKs signaling pathways [23], [54], [55], [56]. In a previous study, apelin stimulations of the PI3K/Akt and/or the MAPK pathways were found to promote cell proliferation, survival, migration and metabolic functions in various cell types [9]. AMPK is another key intracellular effector activated by apelin signaling in skeletal muscle cells, adipocytes and enterocytes. In these cells, apelin-induced AMPK activation mediates different effects, including, the inhibition of lipolysis, glucose uptake, and fatty RSL3 oxidation and the promotion of mitochondrial biogenesis and function [9]. In the present study, suppression of PI3K/Akt or p38 MAPK resulted in apelin dysfunction, suggesting that the PI3K/Akt and p38 MAPK pathways contribute to the regulation of lipid metabolism in Hep3B cells. Moreover, inhibition of PI3K or p38 MAPK blocked the phosphorylation of AMPK. These findings indicate that PI3K/Akt and p38 MAPK activated by apelin-13 are upstream kinases of AMPK. In this study, we found the expression of PPARα was significantly increased by apelin-13. Because PPARα is the key regulatory transcription factor for fatty acids oxidation, PPARα induction by apelin-13 may contribute to the anti-steatosis effect, which is consistent with the induction of PPARα in the fasted state [57]. However, increased PPARα expression was not observed after suppressing AMPK, PI3K, ERK, or p38 MAPK, which confirmed their involvements in the lipolytic effect of apelin-13, especially in the induction of PPARα by apelin-13. This observation suggests apelin-13 represses LXR signaling via PPARα activation, because PPARα activation might suppress LXR-SREBP-1c signaling by preventing LXR/RXR formation [57]. PI3K and AMPK have been reported to play similar roles in apelin signaling in adipocytes. Apelin-13 decreased lipid storage in hypertrophic adipocytes in vitro, possibly by upregulating AQP7 expression through the PI3K signaling pathway [58]. Apelin also stimulates AMPK and glucose uptakes in human adipose tissue [16]. Furthermore, apelin promoted brown adipocyte differentiation by increasing the expressions of brown adipogenic and thermogenic transcriptional factors via the PI3K/Akt and AMPK signaling pathways [59]. In addition, in insulin-resistant 3T3-L1 adipocytes, apelin stimulated glucose uptake through the PI3K/Akt pathway, promoted GLUT4 translocation from cytoplasm to the plasma membrane, and modulated inflammatory responses [60]. Initially, apelin and APJ transcript levels were reported to be barely detectable in liver [61], [62], but later, low levels of apelin and APJ were detected [63]. In the present study, we applied 10nM–1μM apelin on Hep3B cells and 1nM–100nM on primary mouse hepatocytes. Potency of apelin in mouse hepatocytes was 100 times higher than that in human Hep3B cell line. This may imply that human hepatocytes in vivo might have better responsiveness than Hep3B cell line, and because apelin could be released from hepatocytes and Kupffer cells [64], [65], [66], local concentrations of apelin in liver could be high enough to induce anti-steatosis. In the hepatic parenchyma of animals with cirrhosis, APJ was found to be overexpressed, whereas apelin was highly expressed by stellate cells [22]. In rats with cirrhosis, apelin levels were higher than in controls and the mRNA levels of apelin and APJ were four- and 300-fold higher, respectively, in cirrhotic hepatic tissues than in normal controls [22]. APJ was also found to be expressed in hepatocytes of human cirrhotic livers [67], and serum apelin levels showed a significant relationship with chronic liver disease severity [68]. In HepG2 cells, hypoxia, TNF-α and angiotensin II induced the expression of APJ [67], and apelin-APJ signaling stimulated the insulin signaling pathway and ameliorated TNF-α-induced reduced glycogen synthesis [18]. Furthermore, apelin has been suggested to participate in hepatic remodeling and in the initiation of carcinogenesis in chronic hepatitis C patients [69], and thus, it would appear the apelin-APJ system is set to attract more research attention. The present study adds to the previously reported protective functions of apelin and APJ in the background of liver steatosis and suggests they have therapeutic potential.