Novel properties of mature adipocytes in obesity and hyperinsulinemia

Abstract: Adipose tissue expansion and dysfunction, which leads to obesity and related metabolic diseases (e.g., diabetes and hypertension) are currently the most costly challenges for public health, yet the mechanisms underlying adipocyte functional dysregulation have not been fully elucidated . Investigation is largely hindered by the unique technical limitations associated with handling these large, lipid-filled fat cells. New techniques need to be developed in order to better understand the physiology and pathology of adipocytes. In Paper I, by using an adipocyte-specific reporter mouse, we proved that previously reported adipocyte flow cytometry techniques missed the major adipocyte population. Therefore, we defined several crucial cytometer settings required for large cell types that allow us to analyze and sort both white and brown mature adipocytes. This improved strategy is applicable to sort adipocytes based on size without fixation, which greatly facilitates subsequent downstream analyses. In combination with immunostaining, the presented approach can effectively sort UCP1 positive adipocytes from mouse white and brown adipose tissue. Furthermore, we demonstrated a heterogeneous ADRB2 expression pattern in human adipocytes, which confirmed the applicability of our newly developed technique to further explore other aspects of adipocyte identity. In Paper II, we developed a MAAC (membrane mature adipocyte aggregate culture) system as a novel, high-viability model for human mature adipocytes. With a permeable membrane insert sitting on top to facilitate cell aggregation and nutrition access, adipocytes cultured in the MAAC system maintained adipogenic properties, did not dedifferentiate and had reduced hypoxia. This newly developed in vitro system allows us to compare depot-specific adipocyte gene expression, and analyze the crosstalk between adipocytes and macrophages. In particular, we demonstrated that human adipocytes can be transdifferentiated to brown-like adipocytes under the conditions of rosiglitazone stimulation or PGC-1α overexpression. Taken together, we provided a versatile tool for modulating primary adipocytes, opening up numerous downstream applications. In Paper III, we revealed that a large group of mature human adipocytes express an array of cell cycle-specific markers indicative of a cell cycle re-entry profile, and that this is associated with whole-body insulin resistance. We demonstrated that insulin is a critical driver of adipocyte cell cycle re-entry, subsequently making them vulnerable to undergo cellularsenescence. Our data showed that hyperinsulinemia in obese patients is associated with increased p16 and senescence associated β-galactosidase activity in mature adipocytes. Furthermore, we showed that senescent adipocytes are hypertrophic and develop a senescence- associated secretory phenotype (SASP), defined by the secretion of IL-6, IL-8, and MCP1. These findings challenge the dogma that adipocytes permanently exit the cell cycle upon differentiation and reveals cellular senescence as a new mechanism associated with inflammation-related adipocyte pathology. In conclusion, the research within this thesis has provided important techniques for both in vitro adipocyte modulation and high throughput flowcytometric adipocyte analysis, supporting multiple downstream research applications to investigate mechanisms regulating adipocyte physiology and pathology. Furthermore, we demonstrated the phenomenon of cell cycle re- entry and senescence in human mature adipocytes, thereby introducing novel insights into obesity and hyperinsulinemia-induced adipocyte dysfunction, suggesting potential targets for treating obesity-related metabolic diseases.