4F, Supporting Fig. 1D,E). Microarray

data for other STAT3-activating cytokines and growth factors and their receptors showed approximately 2.0- and 4.8-fold increases in average in leukemia inhibitory factor receptor (Lifr) and epidermal cell growth factor receptor (Egfr), respectively, in the ATRA + HFHFr group compared with the HFHFr group (Supporting Table 2). However, because ligands of these receptors exist in ob/ob mice, they were unlikely to be significantly involved in hepatic STAT3 activation. These results suggest that ATRA-induced up-regulation of the short LEPR isoform triggered the activation of leptin signaling, consequently leading to the reversal of leptin resistance. We investigated the involvement of RARα in ATRA action. The Lepra BAY 73-4506 mRNA level in the mouse hepatocyte cell line TLR326 increased as a function of ATRA treatment for up to 12 hours (Fig. 5A). Expression of the short LEPR isoform also increased in a dose-dependent manner at 24 hours (Fig. 5B). As observed in vivo, leptin-induced STAT3 phosphorylation was enhanced by the presence of ATRA (Fig. 5C), suggesting that ATRA-induced LEPRa expression was important check details for the activation of the hepatic leptin-signaling pathway. Nuclear hormone receptors, including RARs, bind to a conserved direct repeat (DR) element when they function as transcription factors. In silico analysis of the mouse Lepr promoter region using the

NHR-scan program31 revealed four putative DR elements (DR1-1, DR1-2, MCE公司 DR3, and DR4) (Supporting Fig. 8A). A chromatin immunoprecipitation assay using anti-RARα antibody demonstrated that RARα constitutively occupied DR1-2 and, to a lesser extent, DR4, although there was slightly decreased RARα binding to DR1-2 in the presence of ATRA (Supporting Fig. 8B). This is in contrast to the retinoic

acid response element of the cytochrome P450 26a1 promoter,32 where ATRA-induced recruitment of RARα was observed. We also performed luciferase reporter assays using Lepr promoter-driven luciferase constructs (Fig. 5D). The mouse Lepr promoter that included DR1-2 responded to ATRA and to the selective RARα/β agonist Am8018 (Fig. 5E). Moreover, DR1-2 enhanced the basal activity of a TATA-like promoter in the presence of retinoids, whereas the inverted DR1-2 sequence showed no such effect (Fig. 5F). Although the possibility of involvement of RARβ remains to be determined, we conclude that retinoids directly regulate Lepr transcription through RARs. Am80 was shown to induce differentiation of acute promyelocytic leukemia cells with greater efficiency than ATRA.18, 19 Thus, the potential of clinical application of Am80 in the treatment of insulin resistance was evaluated in KK-Ay mice. In contrast to ATRA, mice fed an Am80-supplemented diet did not exhibit changes in whole body weight, daily food consumption, or hepatic lipid content (Supporting Fig. 9A-E).