Secretome and glycome of mammalian adipocytes under insulin resistant conditions

Abstract

Insulin resistance defines the metabolic syndrome and precedes type 2 diabetes, thus is considered as the hallmark for type 2 diabetes. Prolonged hyperglycemia and hyperinsulinemia are both required for the development of classical insulin resistance. In recent years, accumulative evidence has established that elevated O-GlcNAc levels via either genetic or pharmacological methods lead to insulin resistance in both cultured adipocytes and animal models. Adipocytes, besides being a major site for energy storage, are endocrine in nature and secrete a variety of proteins (adipocytokines) that can modulate insulin sensitivity, hypertension, food intake, and general energy homeostasis. The link between O-GlcNAc levels, insulin resistance, and adipocytokine secretion is further explored in this dissertation. First, by using immortalized and primary rodent adipocytes, the secreted proteome of differentiated adipocytes under insulin responsive and two insulin resistant conditions is explicitly elucidated via shotgun proteomics leading to the identification of 97 and 203 secreted proteins, respectively. More than 80 N-linked glycosylation sites on adipocytokines released by these adipocytes were also mapped. Moreover, we took a step further to address the quantitative secretome of human primary adipocytes with the same technique. Of the 190 secreted proteins identified, we report 20 up-regulated and 6 down-regulated proteins detected in both insulin resistant conditions. Moreover, we applied glycomic techniques to examine (1) the N-linked sites from the pool of secreted proteins and (2) the characterization and relative abundance of complex N- and O-glycans released from adipocytes exposed to different conditions. We identified 91 Nglycosylation sites on the secreted proteins derived from 51 proteins, as well as 155 and 29 released N- and O-glycans respectively. There were moderate alterations in the observed N- and O-linked glycans under the different conditions considered. In short, our studies have provided a list of adipocytokines whose secreted levels vary under insulin resistant conditions. These proteins bear the potential to be prognostic/diagnostic biomarkers for the detection of metabolic syndrome and type 2 diabetes. Also, comparing the results complied from both classical and non-canonical insulin resistance provides further insights for the role of the O-GlcNAc modification on intracellular proteins in the disease progression of type 2 diabetes.