G-quadruplex (GQ) structures formed from guanine-rich sequences are found throughout the genome and are overrepresented in the promoter regions of some oncogenes, at the telomeric ends of eukaryotic chromosomes, and at the 5'-untranslated regions of mRNA. Interaction of small molecule ligands with GQ DNA is an area of great research interest to develop novel anticancer therapeutics and GQ sensors. In this paper we examine the interactions of TMPyP4, its isomer TMPyP2 (containing N-methyl-2-pyridyl substituents, N-Me-2Py) as well as two metal derivatives ZnTMPyP4 and CuTMPyP4 with GQs formed by dTG and dTGT in 100 mM K or Na conditions. The DNA sequences were chosen to elucidate the effect of the 3'-T on the stabilization effect of porphyrins, binding modes, affinities, and stoichiometries determined via circular dichroism melting studies, UV-vis titrations, continuous variation analysis, and fluorescence studies. Our findings demonstrate that the stabilizing abilities of porphyrins are stronger toward (dTG) as compared to (dTGT) (ΔT is 4.4 vs -6.4 for TMPyP4; 12.7 vs 5.7 for TMPyP2; 16.4 vs 12.1 for ZnTMPyP4; and 1.9 vs -8.4 °C for CuTMPyP4) suggesting that the 3'G-tetrad presents at least one of the binding sites. The binding affinity was determined to be moderate (K ∼ 10-10 μM) with a typical binding stoichiometry of 1:1 or 2:1 porphyrin-to-GQ. In all studies, ZnTMPyP4 emerged as a ligand superior to TMPyP4. Overall, our work contributes to clearer understanding of interactions between porphyrins and GQ DNA.
Objective
Immunoglobulin-like Domain-Containing Receptor 1 (ILDR1) is expressed on nutrient sensing cholecystokinin-positive enteroendocrine cells of the gastrointestinal tract and it has the unique ability to induce fat-mediated CCK secretion. However, the role of ILDR1 in CCK-mediated regulation of satiety is unknown. In this study, we examined the effects of ILDR1 on food intake and metabolic activity using mice with genetically-deleted Ildr1.
Methods
The expression of ILDR1 in murine tissues and the measurement of adipocyte cell size were evaluated by light and fluorescence confocal microscopy. The effects of Ildr1 deletion on mouse metabolism were quantitated using CLAMS chambers and by targeted metabolomics assays of multiple tissues. Hormone levels were measured by ELISA. The effects of Ildr1 gene deletion on glucose and insulin levels were determined using in vivo oral glucose tolerance, meal tolerance, and insulin tolerance tests, as well as ex vivo islet perifusion.
Results
ILDR1 is expressed in a wide range of tissues. Analysis of metabolic data revealed that although Ildr1-/- mice consumed more food than wild-type littermates, they gained less weight on a high fat diet and exhibited increased metabolic activity. Adipocytes in Ildr1-/- mice were significantly smaller than in wild-type mice fed either low or high fat diets. ILDR1 was expressed in both alpha and beta cells of pancreatic islets. Based on oral glucose and mixed meal tolerance tests, Ildr1-/- mice were more effective at lowering post-prandial glucose levels, had improved insulin sensitivity, and glucose-regulated insulin secretion was enhanced in mice lacking ILDR1.
Conclusion
Ildr1 loss significantly modified metabolic activity in these mutant mice. While Ildr1 gene deletion increased high fat food intake, it reduced weight gain and improved glucose tolerance. These findings indicate that ILDR1 modulates metabolic responses to feeding in mice.
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