Examining the regulation of adipocytokine expression during chronic insulin resistance
Hayden, Edith Elizabeth
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Type II diabetes, characterized by hyperglycemia and hyperinsulinemia, results in many costly and debilitating patient complications. A broad range of tissues respond to insulin and help to mediate its effects. In particular, adipose tissue secretes proteins known as adipocytokines, which play an important role in whole body energy homeostasis and have been implicated in the pathogenesis of diabetes. Increased flux through the hexosamine biosynthetic pathway and the corresponding increase in intracellular glycosylation of proteins via O-GlcNAc is sufficient to induce insulin resistance (IR) in multiple systems. Previously, our group used shotgun proteomics to identify rodent adipocytokines whose levels are modulated upon the induction of IR by indirectly and directly modulating O-GlcNAc levels. Since adipocytokines levels are regulated primarily at the level of transcription and O-GlcNAc alters the function of many transcription factors, we hypothesized that elevated O-GlcNAc levels on key transcription factors are modulating adipocytokine expression. Here, we show that upon the elevation of O-GlcNAc levels and the induction of IR in mature 3T3-F442a adipocytes, the transcript levels of multiple adipocytokines, as measured by quantitative RT-PCR, reflect the modulation observed at the protein level. We have gone on to validate the adipocytokine transcript levels in mouse models of diabetes. Using inguinal fat pads from the db/db mouse model and the diet-induced obesity mouse model, we have confirmed that the adipocytokines regulated by O-GlcNAc modulation in cell culture are likewise modulated in the whole animal upon a shift to IR. We find that Sp1 is a common cis-acting element on the promoters of co-regulated genes. Sp1 O-GlcNAc modification increases during IR. As measured by chromatin immunoprecipitation, Sp1 and O-GlcNAc modified proteins are enriched on the LPL and SPARC promoters during insulin resistance. These data lead us to the conclusion that adipocytokine expression is modulated by global O-GlcNAc levels and potentially by Sp1. We go on to characterize the primary human adipocyte secretome in order to identify more adipocytokines whose expression is modulated by O-GlcNAc and find that many of the rodent adipocytokines are likewise regulated by O-GlcNAc levels in homo sapiens.