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In our previous report, we demonstrated the possibility that various regulatory neuropeptides influence feeding behavior in the silkworm, Bombyx mori. Among these feeding-related neuropeptides, short neuropeptide F (sNPF) exhibited feeding-accelerating activity when injected into B. mori larvae. Like other insect sNPFs, the deduced amino acid sequence of the cDNA encoding the sNPF precursor appears to produce multiple sNPF and sNPF-related peptides in B. mori. The presence of three sNPFs, sNPF-1, sNPF-2, and sNPF-3, in the brain of B. mori larvae was confirmed by direct MALDI-TOF mass spectrometric profiling. In addition, all three sNPFs are present in other larval ganglia. The presence of sNPF mRNA in the central nervous system (CNS) was also confirmed by Reverse transcription-polymerase chain reaction. Semi-quantitative analyses of sNPFs in the larval brain using matrix-assisted laser desorption ionization time-of-flight mass spectrometry further revealed that brain sNPF levels decrease in response to starvation, and that they recover with the resumption of feeding. These data suggest that sNPFs were depleted by the starvation process. Furthermore, food deprivation decreased the transcriptional levels of the sNPF receptor (BNGR-A10) in the brain and CNS, suggesting that the sNPF system is dependent on the feeding state of the insect and that the sNPF system may be linked to locomotor activity associated with foraging behavior. Since the injection of sNPFs accelerated the onset of feeding in B. mori larvae, we concluded that sNPFs are strongly related to feeding behavior. In addition, semi-quantitative MS analyses revealed that allatostatin, which is present in the larval brain, is also reduced in response to starvation, whereas the peptide level of Bommyosuppressin was not affected by different feeding states.

Using a chimeric mouse humanized liver model, we provided evidence that human hepatocytes are refractory to the mitogenic effects of rodent constitutive androstane receptor (CAR) activators. To evaluate the functional reliability of this model, the present study examined mitogenic responses to phenobarbital (PB) in chimeric mice transplanted with rat hepatocytes, since rats are responsive to CAR activators. Treatment with 1000 ppm PB for 7 days significantly increased replicative DNA synthesis (RDS) in rat hepatocytes of the chimeric mice, demonstrating that the transplanted hepatocyte model is functionally reliable for cell proliferation analysis. Treatment of humanized CAR and pregnane X receptor (PXR) mice (hCAR/hPXR mice) with 1000 ppm PB for 7 days significantly increased hepatocyte RDS together with increases in several mitogenic genes. Global gene expression analysis was performed with liver samples from this and from previous studies focusing on PB-induced Wnt/β-catenin signaling, and showed that altered genes in hCAR/hPXR mice clustered most closely with liver tumor samples from a diethylnitrosamine/PB initiation/promotion study than with wild type mice. However, different gene clusters were observed for chimeric mice with human hepatocytes for Wnt/β-catenin signaling when compared to those of hCAR/hPXR mice, wild type mice and liver tumor samples. The results of this study demonstrate clear differences in the effects of PB on hepatocyte RDS and global gene expression between human hepatocytes of chimeric mice and hCAR/hPXR mice, suggesting that the chimeric mouse model is relevant to humans for studies on the hepatic effects of rodent CAR activators, whereas the hCAR/hPXR mouse is not.

Phenobarbital (PB) is a nongenotoxic hepatocellular carcinogen in rodents. PB induces hepatocellular tumors by activating the constitutive androstane receptor (CAR). Some previous research has suggested the possible involvement of epigenetic regulation in PB-promoted hepatocellular tumorigenesis, but the details of its molecular mechanism are not fully understood. In the present study, comprehensive analyses of DNA methylation, hydroxymethylation and gene expression using microarrays were performed in mouse hepatocellular adenomas induced by a single 90 mg kg intraperitoneal injection dose of diethylnitrosamine (DEN) followed by 500 ppm PB in the diet for 27 weeks. DNA modification and expression of hundreds of genes are coordinately altered in PB-induced mouse hepatocellular adenomas. Of these, gene network analysis showed alterations of CAR signaling and tumor development-related genes. Pathway enrichment analysis revealed that differentially methylated or hydroxymethylated genes belong mainly to pathways involved in development, immune response and cancer cells in contrast to differentially expressed genes belonging primarily to the cell cycle. Furthermore, overlap was evaluated between the genes with altered expression levels with 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) alterations in mouse hepatocellular adenoma induced by DEN/PB and the genes with altered expression levels in the liver of CD-1 mice or humanized chimeric mice treated with PB for 7 days. With the integration of transcriptomic and epigenetic approaches, we detected candidate genes responsible for early key events of PB-promoted mouse hepatocellular tumorigenesis. Interestingly, these genes did not overlap with genes altered by the PB treatment of humanized chimeric mice, thus suggesting a species difference between the effects of PB in mouse and human hepatocytes.