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

Storing excess energy for future use during starvation is critical for the survival of mammals. Much of this energy is stored in the form of triacylglycerol (TAG) within lipid droplets, which are present most abundantly in adipocytes and which, in turn, accumulate in depots such as subcutaneous and visceral adipose tissue in mice and humans. TAG in lipid droplets is mobilized during starvation by lipase-catalyzed hydrolysis (lipolysis) to release energy in the forms of glycerol and free fatty acids, providing fuel to other cell types such as muscle and liver. Previous work investigating formation of lipid droplets and regulation of lipolysis has elucidated the importance of lipid dropletassociated proteins for these processes ( 1, 2 ). Based on shared sequence homology, one set of lipid droplet proteins is grouped as the perilipin-adipophilin-tail interacting protein 47 (PAT/TIP47) family of proteins ( 3 ). PAT-related proteins are functionally conserved from mammals to lower organisms such as Drosophila and Dictyostelium spp ( 4 ). In Drosophila , two PAT domain proteins are encoded by the Lsdp1 and Lsd2 genes. Drosophila loss-of-function Lsd2 mutants are lean, whereas Lsd2 overexpression causes obesity ( 5 ). In mammals, PAT proteins can be divided into exchangeable TAG-associated PAT proteins (EPATs) or constitutively TAG-associated PAT proteins (CPATs). EPATs include the TIP47/perilipin-3 (PLIN3), S3-12/PLIN4, and Abstract The lipid droplet-associated fat specifi c protein 27 (FSP27) suppresses lipolysis and thereby enhances triglyceride accumulation in adipocytes. We and others have recently found FSP27 to be a remarkably short-lived protein (half-life, 15 min) due to its rapid ubiquitination and proteasomal degradation. Thus, we tested the hypothesis that lipolytic agents such as tumor necrosis factor-␣ (TNF-␣ ) and isoproterenol modulate FSP27 levels to regulate FFA release. Consistent with this concept, we showed that the lipolytic actions of TNF-␣ , interleukin-1 ␤ (IL-1 ␤ ), and IFN-␥ are accompanied by marked decreases in FSP27 expression and lipid droplet size in mouse adipocytes. Similar depletion of FSP27 using short interfering RNA (siRNA) mimicked the lipolysis-enhancing effect of TNF-␣ , while maintaining stable FSP27 levels using expression of hemagglutinin epitopetagged FSP27 blocked TNF-␣ -mediated lipolysis. In contrast, we show the robust lipolytic action of isoproterenol is paradoxically associated with increases in FSP27 levels and a delayed degradation rate corresponding to decreased ubiquitination. This catecholamine-mediated increase in FSP27 abundance, probably a feedback mechanism for restraining excessive lipolysis by catecholamines, is mimicked by forskolin or 8-bromo-cAMP treatment and is prevented by the protein kinase A (PKA) inhibitor KT5720 or by PKA depletion using siRNA. Taken together, these data identify the regulation of FSP27 as an important intermediate in the mechanism of lipolysis in adipocytes in response to TNF-␣ and isoproterenol. -Ranjit, S

supporting

confidence: 79%

Regulation of fat specific protein 27 by isoproterenol and TNF-α to control lipolysis in murine adipocytes

Ranjit

Boutet

Gandhi

et al. 2011

“…Here, we demonstrate that Cideb is also detected in the Golgi apparatus, an important site for VLDL lipidation and maturation. Cideb has been previously shown to localize to the LDs and ER by fl uorescent staining and biochemical fractionation, similar to Cidea and Fsp27 ( 43,44,47,52,53 ). Here, using a sucrose density gradient centrifugation approach, we observed that Cideb is also localized to the Golgi apparatus.…”

Section: Discussionmentioning

confidence: 48%

Opposing roles of cell death-inducing DFF45-like effector B and perilipin 2 in controlling hepatic VLDL lipidation

Zhou

et al. 2012

Self Cite

triglycerides (TAG) and cholesterol and for the development of metabolic disorders, including obesity, diabetes, and hepatic steatosis. It is believed that VLDL assembly and maturation involves two steps ( 1, 2 ). The fi rst step is the formation of lipid-poor pre-VLDL particles involving the cotranslational lipidation of apolipoprotein B (apoB) with a few lipids, aided by microsomal triglyceride transfer protein (MTP) ( 3, 4 ). The second step is the transfer of large quantities of lipids, likely from TAG-rich lipid droplets (LD), to the pre-VLDL particles, converting the pre-VLDL particles into lipid-rich, mature VLDL particles ( 2-5 ). Thus far, the subcellular site of the addition of bulk lipids to pre-VLDL particles, the source of the lipids, and the factors promoting VLDL lipidation remain unclear. The endoplasmic reticulum (ER) has been suggested as the VLDL lipidation site because the majority of TAG is synthesized in the ER, and apoB-100 has been shown to be localized to the ER membrane ( 2, 6, 7 ). Others believe that the pre-VLDL particles are assembled in the ER and then exit, with the fi nal VLDL lipidation and maturation occurring primarily within the Golgi apparatus or post-ER compartments ( 8-11 ). For example, the biochemical separation of the lipoprotein particles from McA-RH7777 cells indicated that mature VLDL particles accumulate in the Golgi but not in the ER fractions ( 11,12 ). In addition, the late addition of bulk lipids to pre-VLDL particles is independent of both MTP activity and new triglyceride synthesis ( 12, 13 ).The availability of neutral lipids and the capacity to transfer TAG to pre-VLDL particles are crucial for VLDL lipidation and maturation. The LD, a subcellular organelle, Abstract Regulation of hepatic very low density lipoprotein (VLDL) assembly and maturation is crucial in controlling lipid homeostasis and in the development of metabolic disorders, including obesity, hepatic steatosis, and insulin resistance. Cideb, a member of cell death-inducing DFF45-like effector (CIDE) protein family, has been previously shown to promote VLDL lipidation and maturation. However, the precise subcellular location of Cideb-mediated VLDL lipidation and the factors modulating its activity remain elusive. In addition to its localization to endoplasmic reticulum (ER) and lipid droplets (LD), we observed that Cideb was also localized to the Golgi apparatus. Mature and lipid-rich VLDL particles did not accumulate in the Golgi apparatus in Cideb ؊ / ؊ livers. Interestingly, we observed that hepatic perilipin 2/adipose differentiation-related protein (ADRP) levels were markedly increased in Cideb ؊ / ؊ mice. Liver-specifi c knockdown of perilipin 2 in Cideb ؊ / ؊ mice resulted in the reduced accumulation of hepatic triglycerides (TAG), increased VLDL-TAG secretion, and the accumulation of mature TAG-rich VLDL in the Golgi apparatus. These data reveal that Cideb and perilipin 2 play opposing roles in controlling VLDL lipidation and hepatic lipid homeostasis. China,

“…Depletion of SREBP-1 had no effect on CIDEC mRNA expression in the presence or absence of insulin (data not shown), suggesting that SREBP-1 is not involved in the regulation of CIDEC expression. It has been shown that CIDEC stabilization is associated with triacylglycerol synthesis and LD formation ( 55 ). The present study suggested that SREBP-1c mediates insulin/JNK2-induced de novo fatty acid synthesis.…”

Section: Discussionmentioning

confidence: 58%

A novel JNK2/SREBP-1c pathway involved in insulin-induced fatty acid synthesis in human adipocytes

Ito¹,

Nagasawa²,

Omae³

et al. 2013

cular disease, nonalcoholic steatohepatitis, nephropathy, and several types of cancer ( 1-3 ). WAT mass is determined by the number and size of adipocytes ( 4, 5 ) regulated by cell differentiation, apoptosis, and lipid droplet (LD) formation (6)(7)(8). Insulin is known to inhibit apoptosis ( 9, 10 ) and increase LD formation ( 11,12 ) in adipocytes. Hyperinsulinemia is associated with weight gain in humans ( 13 ). Insulin signaling in adipocytes is critical for the development of obesity ( 14,15 ). Therefore, it has been suggested that insulin is one of the determinants involved in increasing the WAT mass.Our previous study showed that insulin decreases cell death-inducing DNA fragmentation factor-␣ -like effector (CIDE)A expression and increases CIDEC expression, both of which belong to the CIDE family and play critical roles in apoptosis ( 16,17 ) and LD formation ( 18-21 ), and the differential regulation of these genes is related, at least in part, to insulin-induced anti-apoptosis and LD formation in human adipocytes ( 22 ). Our recent study also showed that insulin regulates CIDEA and CIDEC expression via phosphatidylinositol 3-kinase (PI3K), and regulates expression of each protein via Akt1/2-and c-Jun N-terminal kinase (JNK)2-dependent pathways downstream of PI3K, respectively ( 23 ). These results suggest that insulin regulation of CIDEA and CIDEC contribute separately via different signaling pathways to insulin-induced antiapoptosis and LD formation, leading to increases in the number and size of adipocytes. Adipocyte size is determined by LD formation regulated by glucose uptake, de novo fatty acid synthesis, triacylglycerol synthesis, and lipolysis ( 24,25 ). Previous studies suggested that regulation of CIDEC expression contributes to LD maintenance by modulating lipolysis. However, regulation of other genes Press, March 19, 2013 DOI 10.1194 A novel JNK2/SREBP-1c pathway involved in insulininduced fatty acid synthesis in human adipocytes Abbreviations: ACC1, acetyl-CoA carboxylase 1; ACLY, ATP citrate lyase; CIDE, cell death-inducing DNA fragmentation factor-␣ -like effector; DAPI, 4 ′ ,6 ′ -diamidino-2-phenylindole; Dex, dexamethasone; FAS, fatty acid synthase; IPA, Ingenuity Pathway Analysis; JNK, c-Jun N-terminal kinase; LD, lipid droplet; n-SREBP-1c , nuclear form of sterol regulatory element binding protein-1c; PI3K, phosphatidylinositol 3-kinase; pre-SREBP-1c, precursor form of sterol regulatory element binding protein-1c; siJNK, JNK siRNA; siRNA, small interfering RNA; siSREBP-1, SREBP-1 siRNA; SREBP, sterol regulatory element binding protein; WAT, white adipose tissue. Published, JLR Papers in