br Materials and Methods br Results br

Materials and Methods
Results
Discussion Previous studies have indicated that irisin could improve dyslipidemia and hepatic steatosis in mice (Xiong et al., 2015). The decrement of lipid accumulation in hepatocytes was proposed to result mainly from the suppression of triglyceride synthesis (Park et al., 2015). In cultured AML12 hepatocytes, recombinant irisin significantly reduced the PA-induced lipid accumulation, and inhibited the PA-induced increase in lipogenic markers ACC and FAS at the mRNA and protein levels (Park et al., 2015). Our studies identified suppression of cholesterol synthesis as an alternative pathway for irisin to reduce lipid content in hepatocytes. This conclusion is supported by following observations: 1) Administration of irisin reduced the hepatic lipid contents measured by Oil-red staining in lean and obese mice; 2) In mice and cultured hepatocytes, irisin decreased levels of hepatic cholesterol, while demonstrated no effect on triglyceride contents; 3) Irisin inhibited SREBP2 and its target genes HMGCS2 and HMGCR, a key transcriptional factor and the rate-limiting enzymes for cholesterol synthesis respectively; 4) The effects of irisin on genes related to triglyceride synthesis and β-oxidation were negligible in hepatocytes. There exist two possible explanations for the conflicting observations: different cell models and sources of irisin used. In the studies by Park et al. (Park et al., 2015), AML12 mouse hepatocyte cell line was used. This cell line expresses high levels of human transforming growth factor α (TGFα). Whether the high expression of TGFα in AML12 4 mu accounts for the distinct response to irisin remains to be explored. Theoretically, the preparation of irisin may affect its biological functions. This potential is unlikely because we observed no difference in the suppression of cholesterol synthesis between recombinant irisin-Fc and synthetic irisin. While good progress has been made in identifying the physiological actions of irisin, its receptor and intracellular signaling pathway remains largely unknown. Previous studies have shown that irisin may activate AMPK, PI3K/AKT, or p38 MAPK and ERK (Liu et al., 2015b; Yang et al., 2015; Zhang et al., 2014; Lee et al., 2015) in skeletal muscle cells, hepatocytes and adipocytes. Our studies also suggest the presence of a functional receptor for irisin in hepatocytes. Activation of this receptor by irisin may stimulate AMPK activity, leading to the subsequent suppression of SREBP2 expression and nuclear translocalization. This observation is consistent with previous report demonstrating that AMPK could phosphorylate SREBP2 to reduce SREBP2 precursor cleavage and nuclear translocalization (Liu et al., 2015a). Although activation of AMPK has been demonstrated to suppress hepatic lipid synthesis and to promote fatty acid β-oxidation by phosphorylating SREBP1c (Liu et al., 2015a; Ma et al., 2015; Elhanati et al., 2013; Samovski et al., 2015; Chen et al., 2014; Lee et al., 2012), our studies did not detect consistent change in the levels of triglyceride, SREBP1c and its downstream target genes. A much higher dose of irisin may be required for its effect on triglyceride synthesis. Instead, SREBP2 transcription and nuclear translocalization in hepatocytes were significantly attenuated upon activation of AMPK by irisin. The molecular mechanism underlying the selection of SREBP2 over SREBP1 upon activation of irisin receptor in hepatocytes is currently unknown. Emerging evidence has indicated that cross talks between organs are critical for energy homeostasis. The present studies suggest a direct communication between the skeletal muscle and liver via irisin. Our study also provides significant evidence that hepatocytes are targets for irisin in term of the regulation of hepatic cholesterol metabolism. Conditioned medium from C2C12 myocytes overexpressing FNDC5 significantly attenuated cholesterol synthesis in cultured hepatocytes. Other studies also support the concept that irisin may serve as an important cross-organ messenger linking skeletal muscle with the brain, adipose tissue and the cardiovascular system to integrate the exercise with the physiological activities in these organs (Wrann et al., 2013; Huh et al., 2014; Xiong et al., 2015; Song et al., 2014; Wu et al., 2015; Zhu et al., 2015; Zhang et al., 2015). Importantly, irisin acts in concert with fibroblast growth factor 21 to promote adipocyte browning and thermogenesis in humans (Lee et al., 2014). This result indicates that cross talk between skeletal muscle and adipose tissue may be critical for the control of adiposity. Taken together, all these observations suggest that irisin is an important molecule linking the skeletal muscle with the adipose tissue and liver to integrate lipid metabolism.