Perilipin2 plays a positive role in adipocytes during lipolysis by escaping proteasomal degradation.

Perilipin2 (Plin2), also known as adipose differentiation-related protein (ADRP), or adipophilin, is a member of the PAT family involved in lipid droplet (LD) formation in the liver and peripheral tissues. Although Plin2 was originally identified as a highly expressed gene in adipocytes, its physiological role in mature adipocytes is largely unknown. In this report, we investigated the regulation of Plin2 expression and its function in differentiated adipocytes of mouse embryonic fibroblasts (MEFs). Plin2 mRNA levels increased during adipocyte differentiation whereas protein levels did not. Plin2 was degraded through the ubiquitin-proteasome pathway but was inhibited by lipolytic inducers. Furthermore, lentiviral-mediated Plin2 knockdown attenuated lipolysis in differentiated MEFs in a time-dependent manner. Oleic acid-induced LD formation enhanced Plin2 protein stability when it was localized to LDs. Furthermore, a mutational analysis revealed that the ubiquitination and degradation of Plin2 required both the second and third alanine in the N-terminal region. These results suggest that Plin2 is degraded in the cytosol in its N-terminal amino acid sequence-dependent manner and instead becomes stable when localized on LDs. Our findings highlight the relationship between protein stability and a previously unnoticed function of Plin2 during lipolysis in adipocytes.

PPARγ-induced PARylation promotes local DNA demethylation by production of 5-hydroxymethylcytosine.

Recent studies have shown that DNA demethylation goes through the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) by Tet proteins. However, it is still unclear how the target regions for demethylation are distinguished within their genomic context. Here we show that the nuclear receptor peroxisome proliferator-activated receptor-γ (PPARγ) has the ability to direct local demethylation around its binding sites, the PPAR response elements (PPREs), during adipocyte differentiation. PPARγ is a key regulator of the differentiation process that forms a PPARγ co-activator complex on PPREs and activates the expression of adipocyte-specific genes. The complex is poly(ADP-ribosyl)ated (PARylated) on PPREs, and Tet proteins catalyse the conversion of 5mC to 5hmC locally by their ability to bind to the PAR polymer, thereby inducing region-specific demethylation. Our study demonstrates that a sequence-dependent transcription factor complex can, through its post-translational modification, serve for Tet proteins as a landmark to identify sites of DNA demethylation.

Perilipin-mediated lipid droplet formation in adipocytes promotes sterol regulatory element-binding protein-1 processing and triacylglyceride accumulation.

Sterol regulatory element-binding protein-1 (SREBP-1) has been thought to be a critical factor that assists adipogenesis. During adipogenesis SREBP-1 stimulates lipogenic gene expression, and peroxisome proliferator-activated receptor γ (PPARγ) enhances perilipin (plin) gene expression, resulting in generating lipid droplets (LDs) to store triacylglycerol (TAG) in adipocytes. Plin coats adipocyte LDs and protects them from lipolysis. Here we show in white adipose tissue (WAT) of plin-/- mice that nuclear active SREBP-1 and its target gene expression, but not nuclear SREBP-2, significantly decreased on attenuated LD formation. When plin-/- mouse embryonic fibroblasts (MEFs) differentiated into adipocytes, attenuated LDs were formed and nuclear SREBP-1 decreased, but enforced plin expression restored them to their original state. Since LDs are largely derived from the endoplasmic reticulum (ER), alterations in the ER cholesterol content were investigated during adipogenesis of 3T3-L1 cells. The ER cholesterol greatly reduced in differentiated adipocytes. The ER cholesterol level in plin-/- WAT was significantly higher than that of wild-type mice, suggesting that increased LD formation caused a change in ER environment along with a decrease in cholesterol. When GFP-SREBP-1 fusion proteins were exogenously expressed in 3T3-L1 cells, a mutant protein lacking the S1P cleavage site was poorly processed during adipogenesis, providing evidence of the increased canonical pathway for SREBP processing in which SREBP-1 is activated by two cleavage enzymes in the Golgi. Therefore, LD biogenesis may create the ER microenvironment favorable for SREBP-1 activation. We describe the novel interplay between LD formation and SREBP-1 activation through a positive feedback loop.

The gene expression of the myocardial lipid droplet protein is highly regulated by PPARgamma in adipocytes differentiated from MEFs or SVCs.

We demonstrate that expression of the myocardial lipid droplet protein (MLDP) and ERalpha observed in adipose tissues is undetectable in 3T3-L1 cells but detectable in mouse embryonic fibroblasts (MEFs) and stromal-vascular cells (SVCs) during adipocyte differentiation. MLDP gene expression in MEFs or SVCs is induced by treatment with a PPARgamma agonist or forced expression of PPARgamma, indicating that PPARgamma enhances MLDP expression during adipogenesis. PCR analyses reveal the dual expression of SREBP-1a and SREBP-1c in MEFs and SVCs as well as white adipose tissues unlike the predominant expression of SREBP-1a in 3T3-L1 cells. These results suggest that MEFs and SVCs are useful model cells for examining function of MLDP in lipid droplet formation and adipocyte differentiation.