Role and Regulation of Fat Specific Protein (FSP27) in Lipolysis in 3T3-L1 Adipocytes: A Dissertation

The alarming rate of increase in incidence and prevalence of the type 2 diabetes mellitus has prompted intense research on understanding the pathogenesis of the type 2 diabetes. It is observed that the development of type 2 diabetes is preceded by a state of insulin resistance and obesity. Previous studies have suggested that the obesity induced insulin resistance may be mediated by elevated levels of circulating free fatty acids (FFAs). The increase in circulating levels of FFAs may be contributed by the release of FFAs from stored triglycerides (TG) in adipocytes via lipolysis. It is hypothesized that the decrease in levels of circulating FFAs by sequestration and storage of FFAs in adipocytes may prevent deleterious effects of FFAs on insulin sensitivity. Recently our lab and others have shown that the storage of TG in adipocytes is promoted by a novel protein, Fat Specific Protein 27 (FSP27). Although, these studies also revealed FSP27 to be a lipid droplet associated protein that suppresses lipolysis to enhance TG accumulation in adipocytes, the role of FSP27 in lipolysis remains largely undetermined. Therefore, this study investigates the role and regulation of FSP27 in adipocytes in both the basal state, as well as during lipolysis. The studies presented here show FSP27 to be a remarkably short-lived protein (half-life=15 min) due to its rapid ubiquitination and proteasomal degradation. Thus, I tested the hypothesis that lipolytic agents like the cytokine, TNF-α and the catecholamine isoproterenol modulate FSP27 protein levels to regulate FFA release. Consistent with this concept, TNF-α markedly decreased FSP27 mRNA and protein along with lipid droplet size as it increased lipolysis in cultured adipocytes. Similarly, FSP27 depletion using siRNA mimicked the effect of TNF-α to enhance lipolysis, while maintaining stable FSP27 protein levels by expression of HA epitope-tagged FSP27 blocked TNF-α mediated lipolysis. In contrast, the robust lipolytic action of isoproterenol is paradoxically associated with increases in FSP27 protein and a delayed degradation rate that corresponds to decreased ubiquitination. This catecholamine-mediated increase in FSP27 abundance, probably a feedback mechanism to restrain excessive lipolysis by catecholamines, is mimicked by forskolin or 8-Bromo-cAMP treatment, and prevented by Protein Kinase A (PKA) inhibitor KT5720 or PKA depletion using siRNA. These results show that isoproterenol stabililizes FSP27 via the canonical PKA pathway and increased cAMP levels. However, the work presented here also suggests that FSP27 does not get phosphorylated in response to isoproterenol treatment, and the stabilization of FSP27 is independent of isoproterenol mediated lipolysis. The data presented in this thesis not only identifies the regulation of FSP27 as an important intermediate in mechanism of lipolysis in adipocytes in response to TNF-α and isoproterenol, but also suggests that FSP27 may be a possible therapeutic target to modulate lipolysis in adipocytes

Fat-specific protein 27, a novel lipid droplet protein that enhances triglyceride storage

Fat-specific protein (FSP)27/Cidec is most highly expressed in white and brown adipose tissues and increases in abundance by over 50-fold during adipogenesis. However, its function in adipocytes has remained elusive since its discovery over 15 years ago. Here we demonstrate that FSP27/Cidec localizes to lipid droplets in cultured adipocytes and functions to promote lipid accumulation. Ectopically expressed FSP27-GFP surrounds lipid droplets in 3T3-L1 adipocytes and colocalizes with the known lipid droplet protein perilipin. Immunostaining of endogenous FSP27 in 3T3-L1 adipocytes also confirmed its presence on lipid droplets. FSP27-GFP expression also markedly increases lipid droplet size and enhances accumulation of total neutral lipids in 3T3-L1 preadipocytes as well as other cell types such as COS cells. Conversely, RNA interference-based FSP27/Cidec depletion in mature adipocytes significantly stimulates lipolysis and reduces the size of lipid droplets. These data reveal FSP27/Cidec as a novel adipocyte lipid droplet protein that negatively regulates lipolysis and promotes triglyceride accumulation

Mismatch repair proteins and AID activity are required for the dominant negative function of C-terminally deleted AID in class switching

Activation-induced cytidine deaminase (AID) is essential for class-switch recombination (CSR) and somatic hypermutation (SHM) of Ig genes. The AID C terminus is required for CSR, but not for S-region DNA double-strand breaks (DSBs) during CSR, and it is not required for SHM. AID lacking the C terminus (DeltaAID) is a dominant negative (DN) mutant, because human patients heterozygous for this mutant fail to undergo CSR. In agreement, we show that DeltaAID is a DN mutant when expressed in AID-sufficient mouse splenic B cells. To have DN function, DeltaAID must have deaminase activity, suggesting that its ability to induce DSBs is important for the DN function. Supporting this hypothesis, Msh2-Msh6 have been shown to contribute to DSB formation in S regions, and we find in this study that Msh2 is required for the DN activity, because DeltaAID is not a DN mutant in msh2(-/-) cells. Our results suggest that the DNA DSBs induced by DeltaAID are unable to participate in CSR and might interfere with the ability of full-length AID to participate in CSR. We propose that DeltaAID is impaired in its ability to recruit nonhomologous end joining repair factors, resulting in accumulation of DSBs that undergo aberrant resection. Supporting this hypothesis, we find that the S-S junctions induced by DeltaAID have longer microhomologies than do those induced by full-length AID. In addition, our data suggest that AID binds Smu regions in vivo as a monomer

Cidea is associated with lipid droplets and insulin sensitivity in humans

Storage of energy as triglyceride in large adipose-specific lipid droplets is a fundamental need in all mammals. Efficient sequestration of fat in adipocytes also prevents fatty acid overload in skeletal muscle and liver, which can impair insulin signaling. Here we report that the Cide domain-containing protein Cidea, previously thought to be a mitochondrial protein, colocalizes around lipid droplets with perilipin, a regulator of lipolysis. Cidea-GFP greatly enhances lipid droplet size when ectopically expressed in preadipocytes or COS cells. These results explain previous findings showing that depletion of Cidea with RNAi markedly elevates lipolysis in human adipocytes. Like perilipin, Cidea and the related lipid droplet protein Cidec/FSP27 are controlled by peroxisome proliferator-activated receptor gamma (PPARgamma). Treatment of lean or obese mice with the PPARgamma agonist rosiglitazone markedly up-regulates Cidea expression in white adipose tissue (WAT), increasing lipid deposition. Strikingly, in both omental and s.c. WAT from BMI-matched obese humans, expression of Cidea, Cidec/FSP27, and perilipin correlates positively with insulin sensitivity (HOMA-IR index). Thus, Cidea and other lipid droplet proteins define a novel, highly regulated pathway of triglyceride deposition in human WAT. The data support a model whereby failure of this pathway results in ectopic lipid accumulation, insulin resistance, and its associated comorbidities in humans