Student Theses and Dissertations

Date of Award

2022

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Cohen Paul Laboratory

Abstract

A major threat to human health, obesity is associated with increased risk of cardiometabolic complications such as cardiovascular disease, type 2 diabetes, hypertension, and many types of cancers. Whereas white adipocytes efficiently store energy in the form of triglycerides, thermogenic brown and beige adipocytes can dissipate energy into heat. Adipose tissue is now considered an important endocrine organ, and increasing evidence suggests that divergent metabolic effects of different types of adipocytes are in part mediated by their secretory function. In addition, dysregulation of adipose tissue’s secretory function in the setting of chronic positive energy balance plays a central role in the pathophysiology of obesity and its complications. However, the vast majority of bioactive peptides secreted by adipose tissue, collectively referred to as adipokines, remain uncharacterized. We employed bio-orthogonal non-canonical amino acid tagging (BONCAT) and mass spectrometry to comprehensively characterize the secretome of murine visceral and subcutaneous white adipocytes and interscapular brown adipocytes. Over 600 proteins were identified, the majority of which showed cell type specificity with functional enrichment. BONCAT was also applied in vivo to label and characterize the nascent serum proteome of mice, and bioinformatic analyses suggested that adipose tissue is an important contributor to the serum proteome. We here describe two candidate adipokines identified from these profiling studies, C11orf54 and leucine-rich α-2 glycoprotein 1 (LRG1). C11orf54 was shown to be a cold-induced, brown/beige enriched protein. Despite the lack of a signal peptide, the long isoform (isoform 1) of C11orf54 was validated to be a bona fide secreted factor, while the short isoform (isoform 2) was predominantly intracellular. Functional studies with mice lacking C11orf54 isoform 1 suggest that it may regulate total body energy expenditure. Further studies will be performed to validate and decipher the molecular mechanism underlying this physiological effect. LRG1 was identified as an obesity-regulated adipokine secreted by mature adipocytes. Plasma LRG1 levels were increased in aged and diet-induced obese (DIO) mice. LRG1 overexpression significantly improved glucose and insulin tolerance in DIO mice, despite equivalent body weights. AAV-mediated longitudinal overexpression of LRG1 in the db/db model of type 2 diabetes resulted in accelerated weight gain due to increased white fat mass, but improved insulin tolerance and a delayed diabetic phenotype. This effect was associated with markedly reduced macrophage accumulation in white adipose tissues and a dramatic reduction in systemic inflammation. At the molecular level, LRG1 was shown to bind extracellular cytochrome c (Cyt c), whose levels in serum increased with both diet-induced and genetic obesity. LRG1’s ability to bind Cyt c led to dampening of Cyt c’s pro-inflammatory effect on the macrophage innate immune signaling pathway. These data support a new role for LRG1 as an insulin sensitizer and modulator of inflammation with therapeutic potential. Overall, the work described here provides a thorough characterization of the secretome of white and brown adipocytes, and functional studies on previously undescribed adipokines elucidate novel mechanisms at the intersection of obesity, inflammation, and associated pathology.

Comments

A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

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