Transcriptome analysis of ciliary-dependent MCH signaling in differentiating 3T3-L1 pre-adipocytes.

An understanding of adipocyte responsiveness to G-protein-coupled receptor-(GPCR) derived signals must take into consideration the role of membrane microenvironments; that individual sub-populations of proteins may vary significantly across different regions of the cell, and that cell differentiation alters those microenvironments. 3T3-L1 pre-adipocytes undergo a dramatic phenotypic transformation during differentiation into adipocytes, requiring the development of a transient primary cilium. We demonstrate that melanin-concentrating hormone (MCH) receptor 1, a GPCR that stimulates appetite, translocates to the transient primary cilium during early 3T3-L1 cell adipogenesis. Furthermore, we used RNA-Seq to investigate whether MCH signaling is influenced by its receptor localization and whether MCH can influence the transcriptome of early adipocyte development. We found that MCH signaling is sensitive to receptor localization to cilia, and this alters the adipogenic transcriptional program. Also, novel MCH signaling pathways in 3T3-L1 cells are identified, including those for circadian rhythm, the inflammatory response, and ciliary biogenesis. The presence of active MCH-signaling pathways in pre-adipocytes and the discovery that these pathways intersect with the early adipogenic program, among other newly-identified signaling pathways, suggests that the use of MCH receptor 1 antagonists for clinical interventions may have unintended consequences on adipose tissue development.

Modular Synthesis of Peptide-Based Single- and Multimodal Targeted Molecular Imaging Agents.

A practical, modular synthesis of targeted molecular imaging agents (TMIAs) containing near-infrared dyes for optical molecular imaging (OMI) or chelated metals for magnetic resonance imaging (MRI) and single-photon emission correlation tomography (SPECT) or positron emission tomography (PET) has been developed. In the method, imaging modules are formed early in the synthesis by attaching imaging agents to the side chain of protected lysines. These modules may be assembled to provide a given set of single- or dual-modal imaging agents, which may be conjugated in the last steps of the synthesis under mild conditions to linkers and targeting groups. A key discovery was the ability of a metal such as gadolinium, useful in MRI, to serve as a protecting group for the chelator, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). It was further discovered that two lanthanide metals, La and Ce, can double as protecting groups and placeholder metals, which may be transmetalated under mild conditions by metals used for PET in the final step. The modular method enabled the synthesis of discrete targeted probes with two of the same or different dyes, two same or different metals, or mixtures of dyes and metals. The approach was exemplified by the synthesis of single- or dual-modal imaging modules for MRI-OMI, PET-OMI, and PET-MRI, followed by conjugation to the integrin-seeking peptide, c(RGDyK). For Gd modules, their efficacy for MRI was verified by measuring the NMR spin-lattice relaxivity. To validate functional imaging of TMIAs, dual-modal agents containing Cy5.5 were shown to target A549 cancer cells by confocal fluorescence microscopy.