Anandamide (N‐arachidonoylethanolamine, AEA) or its metabolites participate in energy balance mainly through feeding modulation. In addition, AEA has been found to increase 3T3–L1 adipocyte differentiation process. In this study, the effect of AEA, R(+)‐methanandamide (R(+)‐mAEA), URB597, and indomethacin on primary rat adipocyte differentiation was evaluated by a flow cytometry method and by Oil Red‐O staining. Reverse transcription‐PCR and western blotting analysis were performed in order to study the effect of AEA on peroxisome proliferator–activated receptor (PPAR)γ2, cannabinoid receptors (CBRs), fatty acid amidohydrolase (FAAH), and cyclooxygenase‐2 (COX‐2) expression, during the differentiation process. AEA increased adipocyte differentiation in primary cell cultures in a concentration‐ and time‐dependent manner and induced PPARγ2 gene expression, confirming findings with 3T3–L1 cell line. CB1R, FAAH, and COX‐2 expression was also increased while CB2R expression was decreased. Inhibition of FAAH and COX‐2 attenuated the AEA‐induced differentiation. Our findings indicate that AEA regulates energy homeostasis not only by appetite modulation but may also regulate adipocyte differentiation and phenotype.

It has been shown that gastrin releasing peptide receptors (GRPRs) are overexpressed in various types of cancer cells. Bombesin is an analogue of the mammalian GRP that binds with high specificity and affinity to GRPRs. Significant research efforts have been lately devoted to the design of radiolabeled 8 or 14 aminoacid bombesin (BN) peptides for the detection (either with gamma or positron emitting radionuclides) and therapy (with beta(-) emitting radionuclides) of cancer. The specific aim of the present study was to further investigate the radiolabeled peptide structure and to determine whether the total absence of a linker or the use of a basic diverse amino acid linker could influence the biodistribution profile of the new compounds for specific targeting of human prostate cancer. Thus, two new derivatives with the structure Gly-Gly-Cys-X-BN[2-14], where linker X is either zero (I) or Orn-Orn-Orn (Orn: ornithine) (II) were designed and synthesized. The corresponding (99m)Tc-BN derivatives were obtained with high radiochemical yield (>98%) and had almost identical retention times in RP-HPLC with the (185/187)Re complexes, which were also characterized by ESI-MS. Metabolic stability was found to be high in human plasma, moderate in PC-3 cells, and rather low in mouse liver and kidney homogenates for both BN derivatives studied. The BN derivative without the spacer was less stable in cell culture and liver homogenates. A satisfactory binding affinity to GRPRs, in the nanomolar range, was obtained for both BN derivatives as well as for their Re complexes, with BN (II) demonstrating the highest one. In vitro internalization/externalization assays indicated that approximately 6% of BN (I) and approximately 25% of BN (II) were internalized into PC-3 cells. In vivo evaluation in normal Swiss mice and in tumor bearing SCID mice showed that BN (II) presented higher tumor and pancreas uptake than BN (I). Small animal SPECT dynamic imaging, carried out after an injection of BN (II) in mice bearing PC-3 tumors, resulted in PC-3 tumor delineation with low background activity. Overall, this study performed for two new N(3)S-X-BN[2-14] derivatives indicated that hydrophilicity and charge strongly affected the in vitro and in vivo binding properties and the biodistribution pattern. This finding is confirmed by SPECT imaging of BN (II), which is under further in vivo evaluation for detecting cancer-positive GRPRs.

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