Post-translational Regulation of Adipose Differentiation-related Protein by the Ubiquitin/Proteasome Pathway

The fat-specific protein 27 (Fsp27), a protein localized to lipid droplets (LDs), plays an important role in controlling lipid storage and mitochondrial activity in adipocytes. Fsp27-null mice display increased energy expenditure and are resistant to high fat diet-induced obesity and diabetes. However, little is known about how the Fsp27 protein is regulated. Here, we show that Fsp27 stability is controlled by the ubiquitin-dependent proteasomal degradation pathway in adipocytes. The ubiquitination of Fsp27 is regulated by three lysine residues located in the C-terminal region. Substitution of these lysine residues with alanines greatly increased Fsp27 stability and enhanced lipid storage in adipocytes. Furthermore, Fsp27 was stabilized and rapidly accumulated following treatment with ␤-agonists that induce lipolysis and fatty acid re-esterification in adipocytes. More importantly, Fsp27 stabilization was dependent on triacylglycerol synthesis and LD formation, because knockdown of diacylglycerol acyltransferase in adipocytes significantly reduced Fsp27 accumulation in adipocytes. Finally, we observed that increased Fsp27 during ␤-agonist treatment preferentially associated with LDs. Taken together, our data revealed that Fsp27 can be stabilized by free fatty acid availability, triacylglycerol synthesis, and LD formation. The stabilization of Fsp27 when free fatty acids are abundant further enhances lipid storage, providing positive feedback to regulate lipid storage in adipocytes.Cell death-inducing DNA fragmentation factor-45-like effector (Cide) proteins, including Cidea, Cideb, and fat-specific protein 27 (Fsp27, 3 also known as Cidec in humans), are a family of proteins shown to play critical roles in controlling metabolism homeostasis (1). Our previous work demonstrated that mice with a deficiency in Cidea or Cideb have higher energy expenditure and enhanced insulin sensitivity and are resistant to high fat diet-induced obesity and diabetes (2, 3). Fsp27 is enriched in adipocytes, in both white adipose tissue and brown adipose tissue (2, 4). The Fsp27 protein is detected in the lipid droplet (LD)-enriched fraction (5), and its overexpression can promote triacylglycerol (TAG) storage (6, 7). Interestingly, Fsp27 and Cidea mRNAs have also been detected in fatty livers, where an excess amount of lipids accumulate and large LDs form (8 -10). More recently, Fsp27 was demonstrated to be a direct mediator of peroxisome proliferator-activated receptor ␥-dependent hepatic steatosis (10). In accordance with a role for Fsp27 in LD formation, Fsp27 deficiency results in dramatically reduced white adipose tissue deposits and the acquisition of a brown fat-like morphology in these white adipose tissues, which is characterized by the appearance of smaller LDs and increased mitochondrial size and activity (11, 12). Furthermore, both Fsp27-deficient and Fsp27/leptin double-deficient mice display improved insulin sensitivity and lean phenotype (12). Except for one study showing that Cidea is degraded through the ubiquitin-m...

Section: Fsp27 Is Stabilized By ␤-Agonistsupporting

confidence: 50%

Section: Fsp27 Is Stabilized By ␤-Agonistmentioning

confidence: 68%

Fat-specific Protein 27 Undergoes Ubiquitin-dependent Degradation Regulated by Triacylglycerol Synthesis and Lipid Droplet Formation

Nian

Sun

et al. 2010

“…3). CHO-K1 cells expressing mATGL(S47A)-CFP alone or co-expressing mATGL(S47A)-CFP and CGI-58-YFP were incubated overnight with 400 M oleic acid to increase LD formation and levels of endogenous perilipin 2 on LDs (35). Co-expression of CGI-58-YFP with mATGL(S47A)-CFP increased recruitment of mATGL to LDs, when compared with results obtained in cells expressing only mATGL(S47A)-CFP (Fig.…”

Section: Localization Of Cgi-58 In Cells Expressing Perilipins 1 2 mentioning

confidence: 99%

Unique Regulation of Adipose Triglyceride Lipase (ATGL) by Perilipin 5, a Lipid Droplet-associated Protein

Wang

Bell

Sreenevasan

et al. 2011

Self Cite

226

202

Lipolysis is a critical metabolic pathway contributing to energy homeostasis through degradation of triacylglycerides stored in lipid droplets (LDs), releasing fatty acids. Neutral lipid lipases act at the oil/water interface. In mammalian cells, LD surfaces are coated with one or more members of the perilipin protein family, which serve important functions in regulating lipolysis. We investigated mechanisms by which three perilipin proteins control lipolysis by adipocyte triglyceride lipase (ATGL), a key lipase in adipocytes and non-adipose cells. Using a cell culture model, we examined interactions of ATGL and its co-lipase CGI-58 with perilipin 1 (perilipin A), perilipin 2 (adipose differentiation-related protein), and perilipin 5 (LSDP5) using multiple techniques as follows: anisotropy Forster resonance energy transfer, coimmunoprecipitation, [32 P]orthophosphate radiolabeling, and measurement of lipolysis. The results show that ATGL interacts with CGI-58 and perilipin 5; the latter is selectively expressed in oxidative tissues. Both proteins independently recruited ATGL to the LD surface, but with opposite effects; interaction of ATGL with CGI-58 increased lipolysis, whereas interaction of ATGL with perilipin 5 decreased lipolysis. In contrast, neither perilipin 1 nor 2 interacted directly with ATGL. Activation of protein kinase A (PKA) increased [ 32 P]orthophosphate incorporation into perilipin 5 by 2-fold, whereas neither ATGL nor CGI-58 was labeled under the incubation conditions. Cells expressing both ectopic perilipin 5 and ATGL showed a 3-fold increase in lipolysis following activation of PKA. Our studies establish perilipin 5 as a novel ATGL partner and provide evidence that the protein composition of perilipins at the LD surface regulates lipolytic activity of ATGL. Hydrolysis of triacylglycerol (TAG)3 stored in the lipid droplet (LD) compartment provides a convenient source of cellular fuel for energy production during conditions such as fasting. However, lipolysis can contribute to the build up of toxic lipid intermediates and/or oxidized lipids in pathological conditions when cellular lipid homeostasis is altered, e.g. with obesity (1). Therefore, TAG hydrolysis must be carefully controlled to meet tissue-specific requirements for energy or lipid substrates in both adipose and non-adipose tissues. A better understanding of the mechanisms by which cells control lipid mobilization is needed to design novel approaches for intervention and prevention of the pathophysiological consequences of obesity.During the past 10 years, key players in the lipolytic pathway of adipocytes were identified through the study of transgenic mouse models (2-8). Phenotypic analysis of hormone-sensitive lipase null mice suggested the existence of another lipase (7), leading to the identification of adipose triglyceride lipase (ATGL) (6, 9, 10). Characterization of ATGL null mice helped to establish the respective roles of these two lipases in the lipolytic cascade (7). Complete lipolysis requires three enzyme reactions t...

“…ADFP mainly targets the lipid droplet surface, and unbound ADFP protein is rapidly degraded (20). These differences in lipid droplet binding characteristics and/or stability in aqueous environment may be responsible for the lack of Tip47 overexpression observed in the ADFP null mouse tissues studied (26).…”

Section: Discussionmentioning

confidence: 99%

“…4 On the other hand, we have reported that protection of ADFP from proteosomal degradation with the inhibitor, MG-132, is accompanied by a substantial increase (170%) in lipid content compared with control cells untreated with MG-132, suggesting that ADFP, like perilipin, protects neutral lipids from degradation by lipases (20,21).…”

mentioning

confidence: 99%

Functional Compensation for Adipose Differentiation-related Protein (ADFP) by Tip47 in an ADFP Null Embryonic Cell Line

Sztalryd

Bell

et al. 2006

Self Cite

113

115

Ectopic accumulation of lipid droplets in non-adipose tissues correlates with the degree of insulin resistance in these tissues. Emerging evidence indicates that lipid droplets are specialized organelles that participate in lipid metabolism and intracellular trafficking. These properties are thought to derive from the lipid droplet-associated PAT protein family (perilipin, ADFP, and Tip47). The functions of the ubiquitously distributed adipose differentiation-related protein (ADFP) and Tip47 remain unknown. To evaluate the roles of ADFP and Tip47 in lipid biogenesis and metabolism, ADFP null and wild type (wt) clonal cell lines were established from ADFP null and wt mice, respectively. In ADFP null cells, Tip47 was identified as the sole lipid droplet-associated protein from the PAT family by mass spectroscopy, which was further confirmed by immunoblotting and immunocytochemistry. Following incubation with oleic acid, ADFP null cells were able to form lipid droplets to the same extent as wt cells. No statistical differences between the two cell types were observed in NEFA uptake or lipolysis. Small interference RNAs (siRNAs) against Tip47 were found to down-regulate protein levels for Tip47 by 85%. ADFP null cells treated with Tip47 siRNA retained the ability to form lipid droplets but to a lesser extent and shunted the utilization of exogenously added NEFA from triglycerides to phospholipids. These data support the hypothesis that Tip47 plays an important role in lipid metabolism. Tip47 and ADFP in peripheral tissues may play a critical role in regulating the formation and turnover, and hence metabolic consequences, of ectopic fat.Ectopic fat deposition, the accumulation of lipids in lipid droplets in tissues other than adipose, develops in obese patients and is now recognized as a strong prognostic factor for the development of metabolic syndrome (1-3). The molecular mechanisms regulating the formation and metabolism of lipid droplets in non-adipose tissues and their dysfunction in pathophysiological states are not well understood. Recent proteomic studies indicate that lipid droplets are surrounded by a protein coat that provides an interface for lipid metabolic processes, including transport, lipogenesis, and lipolysis (4 -8). Even more importantly, these studies identify a proteome "signature" for lipid droplets that consistently includes at least one member of the PAT protein family (originally named for perilipin, ADFP and Tip47). A PAT protein is always present and generally represents the most abundant lipid droplet protein, suggesting at least an important structural role for this class of proteins in lipid droplet machinery.The mammalian PAT family includes five members: perilipin, ADFP, 3 tail interacting protein of 47 kDa (Tip47), S3-12, and PAT-1 (9). The PAT proteins are defined by primary sequence homology and are well conserved within the family and across species (10). Recent proteomic studies revealed heterogeneity and tissue-specific differences in droplet-associated proteins (4 -8). PAT prot...