? DESCRIPTION (provided by applicant): The adipokine hormone, leptin informs target cells regarding body fat stores and metabolic status. A key functional response to leptin signaling is an increase in growth hormone (GH)-secretion by pituitary somatotropes. Loss of leptin signaling results in GH deficiency (GHD), with attendant metabolic disorders including adult onset obesity, loss of bone growth in children, low bone density, loss of lean muscle mass and frailty in adults. An understanding of the mechanisms of leptin-induced GH secretion would provide a basis for prevention and correction of these metabolic disorders. The mechanisms mediating leptin signaling to somatotrope GH-secretion are unknown, representing a major knowledge gap that will be addressed in the pilot studies described in this application. To study the mechanisms underlying leptin signaling to somatotrope function, we employed Cre-loxP technology to ablate the murine leptin receptor (floxed alleles of Lepr exon 1) selectively in somatotropes. This model allows us to study the role of leptin signaling specifically in somatotropes in an animal that is otherwise healthy. We have described the adult onset GHD and obesity and consequent metabolic dysfunctions in these mice. We recently introduced a Cre-recombinase reporter transgene into the model, to allow studies of somatotropes identified by fluorescence. The objective of this pilot study is to utilize this newly validated cellular mode to test our central hypothesis that leptin signaling to control somatotrope function is mediated through the attenuation of miRNA- directed, post-transcriptional control of GH mRNA translation and GH protein accumulation. This hypothesis was based on our recently published study indicating that the mutant somatotropes have normal levels of GH mRNA, but reduced GH protein accumulation, suggesting that the deficit in GH secretion is due to de-regulation of mRNA translational control. We have identified binding sites for multiple miRNAs within the GH mRNA 3' untranslated region (3' UTR), 3 of which were specifically elevated in pituitaries from mutant mice. However, we have not proven that these changes are specifically in somatotropes. Thus, the overall aim is to determine if candidate miRNAs are involved in leptin or ghrelin signaling to somatotropes. In Subaim 1, we will use our newly developed model to perform step-wise experiments designed to determine if mutant somatotropes have higher levels of candidate miRNAs. Multiple approaches will validate the short-term culture and FACS. Subaim 2 assesses the efficacy of selective antagomirs in the restoration of GH stores and will also include an unbiased approach to identify the region within the Gh mRNA that mediates translational control. Subaim 3 will determine if Ghrelin restores GH hormone stores in mutant somatotropes by attenuating candidate miRNAs. This pilot study will explore specific mechanisms used by leptin and ghrelin in the optimization of somatotrope function and the prevention of GH deficiency. Antagomir rescue experiments may lead to future therapeutic approaches for GHD.

R03HD082793

2015-07-10

2018-06-30