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Adipose tissue is a key regulator of energy metabolism and glucose homeostasis by promoting triglyceride storage and breakdown in various physiological states. Obesity, however, alters adipose tissue metabolism, inducing chronic inflammation, followed by excessive lipolysis. This results in higher systemic free fatty acid (FFA) levels, leading to desensitization of insulin signaling and ultimately to insulin resistance. Although the link between obesity and progression of insulin resistance and type 2 diabetes mellitus (T2DM) remains unclear, tumor necrosis factor-alpha (TNF-alpha) has been proposed to be a key player in promoting obesity-related development of T2DM through chronic inflammation of adipose tissue. TNF-alpha has direct and indirect mechanisms by which it elicits insulin resistance in adipocytes. TNF-alpha attenuates insulin signaling by directly inhibiting insulin-stimulated tyrosine phosphorylation of the insulin receptor and insulin receptor substrate-1 (IRS-1). Indirectly, TNF-alpha activates signaling pathways to increase lipolysis and FFA release into circulation, leading to insulin resistance. Lipid droplet-associated fat specific protein 27 (FSP27) protects adipocytes from lipolysis by regulating the lipolytic capacity as well as transcription of adipose triglyceride lipase (ATGL). It has been observed that TNF-alpha promotes lipolysis by reducing the expression of FSP27 in murine adipocytes. The effect of TNF-alpha on lipolysis human adipocytes has also been studied; yet its effect on promoting insulin resistance in human adipocytes still remains elusive. In the present study, we examined the effect of FSP27 on TNF-alpha induced lipolysis and insulin resistance in human adipocytes. TNF-alpha enhanced lipolysis in cultured human adipocytes. In addition, TNF-alpha reduced the expression of endogenous FSP7 and the phosphorylation of AKT, inhibiting the activation of insulin signaling pathway in cultured human adipocytes. FSP27 overexpression, however, attenuated TNF-alpha induced lipolysis and restored activation of insulin signaling through phosphorylation of AKT in cultured human adipocytes. Taken together, these data suggest that FSP27 has a protective effect against TNF-alpha induced lipolysis and insulin resistance through regulating lipolysis and insulin signaling in human adipocytes.

Obesity is a rapidly growing epidemic in the USA and worldwide. While the molecular and cellular mechanism of obesity is incompletely understood, studies have shown that excess adiposity may arise from increased adipogenesis (hyperplasia) and adipocyte size (hypertrophy) . Emerging evidence underscores autophagy as an important mediator of adipogenesis and adiposity. We are interested in the upstream regulator of adipocyte autophagy and how it impacts adipocyte biology. Given that metabolic stress activates transcription factor FoxO1 in obesity, my dissertation project is designed to depict the role of FoxO1 in adipocyte autophagy and biology. We found that FoxO1 upregulation was concomitant with elevation of autophagy activity during adipogenesis. Inhibition of FoxO1 suppressed autophagy flux and almost completely prevented adipocyte differentiation. For the first time, we found that the kinetics of FoxO1 activation followed a series of sigmoid curves that showed multiple activation-inactivation transitions during adipogenesis. Our study provides critical evidence casting light on the controversy in the literature that either persistent inhibition or activation of FoxO1 suppresses adipogenesis. In addition, we identified two central pathways that FoxO1-mediated autophagy regulated adipocyte biology: (1) to control lipid droplet growth via fat specific protein 27 (FSP27) in adipocytes; and (2) to differentially regulate mitochondrial uncoupling proteins (UCP) that have been implicated in browning of white adipose tissue and redox homeostasis. Mechanistically, FoxO1 appears to induce autophagy through the transcription factor EB (Tfeb), which was previously shown to regulate both autophagosome and lysosome. Chromatin immunoprecipitation assay demonstrated that FoxO1 directly bound to the promoter of Tfeb, and inhibition of FoxO1 attenuated the binding, which resulted in reduced Tfeb expression. To investigate the role of FoxO1 in vivo, we have developed mouse models to modulate FoxO1 in adipose tissue using an inducible Cre-loxP system. Tamoxifen is widely used to activate the inducible Cre recombinase that spatiotemporally control target gene expression in animal models, but it was unclear whether tamoxifen itself may affect adiposity and confounds phenotyping. Part of my dissertation work was to address this important question. We found that tamoxifen led to reduced fat mass independent of Cre, which lasted for 4-5 weeks. Mechanistically, Tamoxifen induced reactive oxygen species (ROS) and augmented apoptosis. Our data reveals a critical period of recovery following tamoxifen treatment in the study of inducible knockout mice. Together, my dissertation work demonstrates FoxO1 as a critical regulator of adipocyte autophagy via Tfeb during adipogenesis. FoxO1-mediated autophagy controls FSP27, lipid droplet growth, and mitochondrial uncoupling proteins. Further study of FoxO1-autophagy axis in obese subjects is of physiological significance, and the investigation is under way. / Ph. D. / Obesity incidence is rapidly growing in the USA and worldwide. The mechanism of obesity is incompletely understood at present. My dissertation project was designed to address the cellular aspect of obesity. The data suggest that FoxO1, a molecule that can regulate gene expression, controls fat cell formation and expansion, both of which have been shown to increase fat mass in obese individuals. My research also indicates that FoxO1 regulates the ability of fat cells to store lipids and expend energy in the form of heat. Mechanistic studies show that FoxO1 exerts the above mentioned functions by mediating autophagy, a process that plays important roles in cellular component recycling and modeling. To validate these findings in a more physiological setting, our research team and I have started to generate mouse model and study how the modulation of FoxO1 and autophagy may affect fat mass and energy expenditure. This exciting work is under way.

In this study, I examined the use of mouse (Mus musculus) Fat Specific Protein 27 (FSP27) ectopically expressed in Arabidopsis thaliana and Nicotiana benthamiana as a means to increase lipid droplet (LD) presence in plant tissues. In mammalian cells, this protein induces cytoplasmic LD clustering and fusion and helps prevent breakdown of LDs contributing to the large, single LD that dominates adipocytes. When expressed in Arabidopsis thaliana and Nicotiana benthamiana, FSP27 retained its functionality and supported the accumulation of numerous and large cytoplasmic LDs, although it failed to produce the large, single LD that typifies adipose cells. FSP27 has no obvious homologs in plants, but a search for possible distant homologs in Arabidopsis returned a Tudor/PWWP/MBT protein coded for by the gene AT1G80810 which for the purposes of this study, we have called LIPID REGULATORY TUDOR DOMAIN CONTAINING GENE 1 (LRT1). As a possible homolog of FSP27, LRT1 was expected to have a positive regulatory effect on LDs in cells. Instead, a negative regulatory effect was observed in which disruption of the gene induced an accumulation of cytoplasmic LDs in non-seed tissue. A study of lrt1 mutants demonstrated that disruption this gene is the causal factor of the cytoplasmic LD accumulation observed in the mutants, that this phenotype occurs in above ground tissues and is present throughout the early growth stages of the plant. Further examination of lrt1 mutant plants has allowed a preliminary understanding of the role LRT1 may play in LD regulation. Taken together, the results of this study point towards some promising strategies to increase LD content in plant tissues.