0 ± 0.15; HFHFr, 1.6 ± 0.29; ATRA + HFHFr, 2.1 ± 0.17), albeit not to a statistically significant degree.

These results suggest that ATRA also normalizes insulin sensitivity in the diet-induced NAFLD mice, possibly by reversing leptin resistance. We examined retinoid signaling in the livers of NAFLD mice, in which ligand-dependent RAR-mediated transcriptional regulation plays a central role. Although hepatic RARα expression was not changed, expression of the RARα target gene Rarb was significantly down-regulated in HFHFr mice, whereas ATRA reversed this effect (Supporting Fig. 6A-C). This result was consistent with our previous observation that hepatic retinoid signaling is impaired in NAFLD patients22 check details and

prompted us to perform transcriptome analysis using complementary DNA microarrays. By identifying genes with elevated expression in the ATRA + HFHFr group compared with the HFHFr group, four independent probes corresponding to Lepr were ranked as the highest 10 (Table 1). This finding was consistent with the leptin-dependent effect of ATRA on insulin sensitivity. qPCR confirmed significant up-regulation of LEPRa as well as IGF binding protein 2 (IGFBP2), which is expressed in the liver in response to systemic leptin administration29 (Supporting Fig. 6D,E). This finding suggests that the leptin-signaling pathway was activated in the livers of mice fed the ATRA + HFHFr Mdm2 inhibitor diet. We next examined the expression of leptin-signaling proteins. Consistent with

the qPCR data, Western blotting revealed that the expression of the short LEPR isoform was also significantly higher in the ATRA + HFHFr mice compared with that in the HFHFr MCE公司 group (Fig. 4E, Supporting Fig. 5C). Note that LEPRb protein expression was not detected in liver samples. Although the total JAK2 level was lower in HFHFr and ATRA + HFHFr group mice than in controls, its phosphorylation was significantly elevated in the latter group (Fig. 4E, Supporting Fig. 5D,E). Total and phosphorylated STAT3 expression were significantly up-regulated by ATRA, whereas suppressor of cytokine signaling 3 (SOCS3) was not (Fig. 4E, Supporting Fig. 5F-H). STAT3 activation in hepatocytes by ATRA was also demonstrated by immunohistochemistry, showing intense nuclear staining of STAT3 in hepatocytes throughout the liver lobule in the ATRA + HFHFr group, while STAT3 was distributed diffusely in the cytoplasm and nucleus of pericentral hepatocytes in the control and HFHFr groups (Supporting Fig. 7). No changes were observed in the levels of other leptin signaling molecules, adenosine monophosphate-activated protein kinase α, or extracellular signal-regulated kinases 1/29, 30 (data not shown).