Alan R. Kimmel

Perilipin ablation results in a lean mouse with aberrant adipocyte lipolysis, enhanced leptin production, and resistance to diet-induced obesity

Perilipin coats the lipid droplets of adipocytes and is thought to have a role in regulating triacylglycerol hydrolysis. To study the role of perilipin in vivo, we have created a perilipin knockout mouse. Perilipin null (peri−/−) and wild-type (peri+/+) mice consume equal amounts of food, but the adipose tissue mass in the null animals is reduced to ≈30% of that in wild-type animals. Isolated adipocytes of perilipin null mice exhibit elevated basal lipolysis because of the loss of the protective function of perilipin. They also exhibit dramatically attenuated stimulated lipolytic activity, indicating that perilipin is required for maximal lipolytic activity. Plasma leptin concentrations in null animals were greater than expected for the reduced adipose mass. The peri−/− animals have a greater lean body mass and increased metabolic rate but they also show an increased tendency to develop glucose intolerance and peripheral insulin resistance. When fed a high-fat diet, the perilipin null animals are resistant to diet-induced obesity but not to glucose intolerance. The data reveal a major role for perilipin in adipose lipid metabolism and suggest perilipin as a potential target for attacking problems associated with obesity.

Perilipin A is essential for the translocation of hormone-sensitive lipase during lipolytic activation

Akey step in lipolytic activation of adipocytes is the translocation of hormone-sensitive lipase (HSL) from the cytosol to the surface of the lipid storage droplet. Adipocytes from perilipin-null animals have an elevated basal rate of lipolysis compared with adipocytes from wild-type mice, but fail to respond maximally to lipolytic stimuli. This defect is downstream of the β-adrenergic receptor–adenylyl cyclase complex. Now, we show that HSL is basally associated with lipid droplet surfaces at a low level in perilipin nulls, but that stimulated translocation from the cytosol to lipid droplets is absent in adipocytes derived from embryonic fibroblasts of perilipin-null mice. We have also reconstructed the HSL translocation reaction in the nonadipocyte Chinese hamster ovary cell line by introduction of GFP-tagged HSL with and without perilipin A. On activation of protein kinase A, HSL-GFP translocates to lipid droplets only in cells that express fully phosphorylatable perilipin A, confirming that perilipin is required to elicit the HSL translocation reaction. Moreover, in Chinese hamster ovary cells that express both HSL and perilipin A, these two proteins cooperate to produce a more rapidly accelerated lipolysis than do cells that express either of these proteins alone, indicating that lipolysis is a concerted reaction mediated by both protein kinase A–phosphorylated HSL and perilipin A.

Functional Conservation for Lipid Storage Droplet Association among Perilipin, ADRP, and TIP47 (PAT)-related Proteins in Mammals,Drosophila, and Dictyostelium

Intracellular neutral lipid storage droplets are essential organelles of eukaryotic cells, yet little is known about the proteins at their surfaces or about the amino acid sequences that target proteins to these storage droplets. The mammalian proteins Perilipin, ADRP, and TIP47 share extensive amino acid sequence similarity, suggesting a common function. However, while Perilipin and ADRP localize exclusively to neutral lipid storage droplets, an association of TIP47 with intracellular lipid droplets has been controversial. We now show that GFP-tagged TIP47 co-localizes with isolated intracellular lipid droplets. We have also detected a close juxtaposition of TIP47 with the surfaces of lipid storage droplets using antibodies that specifically recognize TIP47, further indicating that TIP47 associates with intracellular lipid storage droplets. Finally, we show that related proteins from species as diverse asDrosophila and Dictyostelium can also target mammalian or Drosophila lipid droplet surfaces in vivo. Thus, sequence and/or structural elements within this evolutionarily ancient protein family are necessary and sufficient to direct association to heterologous intracellular lipid droplet surfaces, strongly indicating that they have a common function for lipid deposition and/or mobilization.

Adoption of PERILIPIN as a unifying nomenclature for the mammalian PAT-family of intracellular lipid storage droplet proteins

The PAT family of proteins has been identified in eukaryotic species as diverse as vertebrates, insects, and amebazoa. These proteins share a highly conserved sequence organization and avidity for the surfaces of intracellular, neutral lipid storage droplets. The current nomenclature of the various members lacks consistency and precision, deriving more from historic context than from recognition of evolutionary relationship and shared function. In consultation with the Mouse Genomic Nomenclature Committee, the Human Genome Organization Genomic Nomenclature Committee, and conferees at the 2007 FASEB Conference on Lipid Droplets: Metabolic Consequences of the Storage of Neutral Lipids, we have established a unifying nomenclature for the gene and protein family members. Each gene member will incorporate the root term PERILIPIN (PLIN), the founding gene of the PAT family, with the different genes/proteins numbered sequentially.

GSK3, a master switch regulating cell-fate specification and tumorigenesis.

Until recently, protein kinase GSK3 (glycogen synthase kinase 3), an essential component for cell-fate specification, had been considered a constitutively activated enzyme subject to developmentally regulated inhibition through hierarchical, linear signaling paths. Data from various systems now indicate more complex scenarios involving activating as well as inhibiting circuits, and the differential formation of multi-protein complexes that antagonistically affect GSK3 function.

On the Control of Lipolysis in Adipocytes

ABSTRACT: The lipolytic reaction in adipocytes is one of the most important reactions in the management of bodily energy reserves, and dysregulation of this reaction may contribute to the symptoms of Type 2 diabetes mellitus. Yet, progress on resolving the molecular details of this reaction has been relatively slow. However, recent developments at the molecular level begin to paint a clearer picture of lipolysis and point to a number of unanswered questions. While HSL has long been known to be the rate‐limiting enzyme of lipolysis, the mechanism by which HSL attacks the droplet lipids is not yet firmly established. Certainly, the immunocytochemical evidence showing the movement of HSL to the lipid droplet upon stimulation leaves little doubt that this translocation is a key aspect of the lipolytic reaction, but whether or not HSL phosphorylation contributes to the translocation, and at which site(s), is as yet unresolved. It will be important to establish whether there is an activation step in addition to the translocation reaction. The participation of perilipin A is indicated by the findings that this protein can protect neutral lipids within droplets from hydrolysis, but active participation in the lipolytic reaction is yet to be proved. Again, it will be important to determine whether mutations of serine residues of PKA phosphorylation sites of perilipins prevent lipolysis, and whether such modifications abolish the physical changes in the droplet surfaces that accompany lipolysis.

Post-translational regulation of adipose differentiation-related protein by the ubiquitin/proteasome pathway.

Adipose differentiation-related protein (ADRP) is localized to lipid droplets in most mammalian cells. ADRP, proposed to regulate fatty acid mobilization and lipid droplet formation, is linked to lipid accumulation in foam cells of human atherosclerotic lesions. In this report, we show that ADRP protein accumulates in Chinese hamster ovary fibroblastic cells cultured in the presence of oleic acid but is destabilized when fatty acid sources are removed from culture serum. The latter effect was blocked by the proteasome inhibitor MG132, whereas inhibitors of other proteolytic processes were ineffective. Pulse-chase experiments confirmed that ADRP degradation is inhibited by MG132. Conditions that stimulate ADRP degradation also promoted the covalent modification of ADRP by ubiquitin, whereas the addition of oleic acid to culture media, which promotes triacylglycerol deposition, blunted the appearance of ubiquitinated-ADRP. Treatment with MG132 increased the levels of ADRP associated with lipid droplets, as well as throughout the cytosol. Finally, we demonstrate that the disappearance of ADRP protein after the onset of perilipin expression during adipocyte differentiation is due to degradation by proteasomes Thus, proteolytic degradation of ADRP mediated through the ubiquitin/proteasome pathway appears to be a major mode for the post-translational regulation of ADRP.

The central role of perilipin a in lipid metabolism and adipocyte lipolysis.

The related disorders of obesity and diabetes are increasing to epidemic proportions. The role of neutral lipid storage and hydrolysis, and hence the adipocyte, is central to understanding this phenomenon. The adipocyte holds the major source of stored energy in the body in the form of triacylglycerols (TAG). It has been known for over 35 years that the breakdown of TAG and release of free (unesterified) fatty acids and glycerol from fat tissue can be regulated by a cAMP‐mediated process. However, beyond the initial signaling cascade, the mechanistic details of this lipolytic reaction have remained unclear. Work in recent years has revealed that both hormone‐sensitive lipase (HSL), generally thought to be the rate‐limiting enzyme, and perilipin, a lipid droplet surface protein, are required for optimal lipid storage and fatty acid release. There are multiple perilipin proteins encoded by mRNA splice variants of a single perilipin gene. The perilipin proteins are polyphosphorylated by protein kinase A and phosphorylation is necessary for translocation of HSL to the lipid droplet and enhanced lipolysis. Hence, the surface of the lipid storage droplet has emerged as a central site of regulation of lipolysis. This review will focus on adipocyte lipolysis with emphasis on hormone signal transduction, lipolytic enzymes, the lipid storage droplet, and fatty acid release from the adipocyte. IUBMB Life, 56: 379‐385, 2004

The murine perilipin gene: the lipid droplet-associated perilipins derive from tissue-specific, mRNA splice variants and define a gene family of ancient origin

Abstract. The Perilipins are a family of intracellular neutral lipid droplet storage proteins that are responsive to acute protein kinase A-mediated, hormonal stimulation. Perilipin (Peri) expression appears to be limited to adipocytes and steroidogenic cells, in which intracellular neutral lipid hydrolysis is regulated by protein kinase A. We have isolated cDNA sets and overlapping genomic fragments of the murine Peri locus and mapped chromosomal location, transcription start sites, polyadenylylation sites, and intron/exon junctions. Data confirm that the Perilipins are encoded by a single-copy gene, with alternative and tissue-specific, mRNA splicing and polyadenylylation yielding four different protein species. The Perilipin proteins have identical ∼22-kDa amino termini with distinct carboxyl terminal sequences of varying lengths. These genomic and transcriptional maps of murine Perilipin are also essential for evaluating presumptive endogenous and targeted mutations within the locus. The N-terminal identity region of the Perilipins defines a sequence motif, which we term PAT, that is shared with the ADRP and TIP47 proteins; additionally, the PAT domain may represent a novel, conserved pattern for lipid storage droplet (LSD) proteins of vertebrates and invertebrates alike. Comparative genomics suggest the presence of related LSD genes in species as diverse as Drosophila and Dictyostelium.

THE NOVEL TYROSINE KINASE ZAK1 ACTIVATES GSK3 TO DIRECT CELL FATE SPECIFICATION

Inhibition of GSK3 by 7-TM Wnt/wg receptor signaling is critical for specifying embryonic cell fate patterns. In Dictyostelium, the 7-TM cAMP receptors regulate GSK3 by parallel, antagonistic pathways to establish a developmental body plan. We describe here a novel tyrosine kinase, ZAK1, downstream of 7-TM cAMP receptor signaling that is required for GSK3 activation during development. zak1-nulls have reduced GSK3 activity and are defective in GSK3-regulated developmental pathways. Moreover, recombinant ZAK1 phosphorylates and activates GSK3 in vitro. We propose that ZAK1 is a positive regulator of GSK3 activity required for cell pattern formation in Dictyostelium and speculate that similar mechanisms exist to antagonize Wnt/wg signaling for metazoan cell fate specification.