Student Theses and Dissertations

Date of Award

2009

Document Type

Thesis

RU Laboratory

Muir Laboratory

Keywords

leptin biodistribution, megalin, positron emission tomography (PET)

Abstract

Leptin is a polypeptide hormone, secreted principally by adipose tissue, which functions as an afferent signal in a feedback loop to maintain body weight and energy homeostasis. In addition to its well documented effects on food intake and energy expenditure, leptin modulates the function of many other physiological systems in mammals, through actions in the central nervous system and periphery. Remarkably, despite extensive studies on leptin receptor expression, the physiological biodistribution of the hormone remains essentially unknown. In order to characterize the distribution leptin in mammals, we have developed methodologies to radiolabel the hormone and visualize its biodistribution using positron emission tomography (PET). Two complementary techniques were developed to label leptin using the positron emitting isotopes 68Ga and 18F. 68Ga labeling was accomplished by lysine-directed conjugation with the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) followed by chelation of the isotope. 18F labeling was accomplished using a site-specific, two step labeling procedure in which an aminooxy moiety was introduced at the Cterminus of leptin using expressed protein ligation (EPL), which was subsequently derivitized with 18F-4-fluorobenzaldehyde in an aniline accelerated radiochemical oximation reaction. These probes were used for PET imaging in mice, rats, and in rhesus macaques. PET imaging in these organisms revealed that the hormone was rapidly taken up by the cortex of the kidney, bone marrow, and visceral organs. Uptake in the kidney was partially saturable with cold ligand, and was not mediated by leptin receptor (ObR). Subsequent analysis in with a kidney specific knockout of the multiligand endocytic transporter megalin revealed loss of leptin in the urine, which was confirmed using PET imaging. Thus, megalin is required for the uptake of leptin in the proximal convoluted tubule within the cortex of the kidney. Subsequent biodistribution experiments revealed that the hormone was taken up by the brain, spleen, liver, fat, and lungs in mice, and that this uptake was leptin receptor dependant. Furthermore, PET imaging in rhesus macaques revealed that leptin was absorbed by the bone marrow and liver. Thus, leptin may activate and modulate hematopoiesis by direct action on either hematopoietic precursors or the stromal support tissue. Thus, novel hysiologically significant interactions of the hormone leptin were revealed using PET.

Comments

A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy.

Permanent URL

http://hdl.handle.net/10209/410

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