Perilipin A is essential for the translocation of hormone-sensitive lipase during lipolytic activation. J Cell Biol

A key 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 -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 A 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

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 regulatefatty acid mobilization and lipid droplet formation, is linked to lipid accumulation in foam cells of human atheroscleroticlesions. In this report, we show that ADRP protein accumulates in Chinese hamster ovary fibroblastic cells cultured in thepresence of oleic acid but is destabilized when fatty acid sources are removed from culture serum. The latter effect was blockedby the proteasome inhibitor MG132, whereas inhibitors of other proteolytic processes were ineffective. Pulse-chase experimentsconfirmed that ADRP degradation is inhibited by MG132. Conditions that stimulate ADRP degradation also promoted the covalentmodification 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 perilipinexpression during adipocyte differentiation is due to degradation by proteasomes Thus, proteolytic degradation of ADRP mediatedthrough the ubiquitin/proteasome pathway appears to be a major mode for the post-translational regulation of ADRP

Distinct cellular pools of perilipin 5 point to roles in lipid trafficking

The PAT family of lipid storage droplet proteins comprised five members, each of which has become an established regulator of cellular neutral lipid metabolism. Perilipin 5 (also known as lsdp-5, MLDP, PAT-1, and OXPAT), the most recently discovered member of the family, has been shown to localize to two distinct intracellular pools: the lipid storage droplet (LD), and a poorly characterized cytosolic fraction. We have characterized the denser of these intracellular pools and find that a population of perilipin 5 not associated with large LDs resides in complexes with a discrete density (~1.15 g/ml) and size (~575 kDa). Using immunofluorescence, western blotting of isolated sucrose density fractions, native gradient gel electrophoresis, and co-immunoprecipitation, we have shown that these small (~15 nm), perilipin 5-encoated structures do not contain the PAT protein perilipin 2 (ADRP), but do contain perilipin 3 and several other as of yet uncharacterized proteins. The size and density of these particles as well as their susceptibility to degradation by lipases suggest that like larger LDs, they have a neutral lipid rich core. When treated with oleic acid to promote neutral lipid deposition, cells ectopically expressing perilipin 5 experienced a reorganization of LDs in the cell, resulting in fewer, larger droplets at the expense of smaller ones. Collectively, these data demonstrate that a portion of cytosolic perilipin 5 resides in high density lipid droplet complexes that participate in cellular neutral lipid accumulation

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 ratelimiting 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

Foundational Concepts and Underlying Theories for Majors in Biochemistry and Molecular Biology

Over the past two years, through an NSF RCN UBE grant, the ASBMB has held regional workshops for faculty members and science educators from around the country that focused on identifying: 1) core principles of biochemistry and molecular biology, 2) essential concepts and underlying theories from physics, chemistry, and mathematics, and 3) foundational skills that undergraduate majors in biochemistry and molecular biology must understand to complete their major coursework. Using information gained from these workshops, as well as from the ASBMB accreditation working group and the NSF Vision and Change report, the Core Concepts working group has developed a consensus list of learning outcomes and objectives based on five foundational concepts (evolution, matter and energy transformation, homeostasis, information flow, and macromolecular structure and function) that represent the expected conceptual knowledge base for undergraduate degrees in biochemistry and molecular biology. This consensus will aid biochemistry and molecular biology educators in the development of assessment tools for the new ASBMB recommended curriculum. © 2013 by The International Union of Biochemistry and Molecular Biology, 00(00):000-000, 2013

PAT proteins, an ancient family of lipid droplet proteins that regulate cellular lipid stores. Biochim Biophys Acta

The PAT family of lipid droplet proteins includes 5 members in mammals: perilipin, adipose differentiation-related protein (ADRP), tail-interacting protein of 47 kiloDaltons (TIP47), S3-12, and OXPAT. Members of this family are also present in evolutionarily distant organisms, including insects, slime molds and fungi. All PAT proteins share sequence similarity and the ability to bind intracellular lipid droplets, either constitutively or in response to metabolic stimuli, such as increased lipid flux into or out of lipid droplets. Positioned at the lipid droplet surface, PAT proteins manage access of other proteins (lipases) to the lipid esters within the lipid droplet core and can interact with cellular machinery important for lipid droplet biogenesis. Genetic variations in the gene for the best characterized of the mammalian PAT proteins, perilipin, have been associated with metabolic phenotypes, including type 2 diabetes mellitus and obesity. In this review, we discuss how the PAT proteins regulate cellular lipid metabolism both in mammals and in model organisms

Cost-Effective Engineering of a Small-Scale Bioreactor

Several methods exist for increasing the scale of cell culture in the laboratory. While these methods provide significant increases in biomass, they are often prohibitively expensive for many laboratories. We have engineered a small-scale bioreactor with a novel means of introducing oxygen through the catalytic breakdown of hydrogen peroxide using a manganese oxide catalyst. We have also adapted and modified an existing assay for dissolved oxygen to be compatible with culture conditions. In this system we have been able to culture CHO cells at densities of up to 107 cells/mL without the use of automated feedback systems

PAT proteins, an ancient family of lipid droplet proteins that regulate cellular lipid stores.

The PAT family of lipid droplet proteins includes 5 members in mammals: perilipin, adipose differentiation-related protein (ADRP), tail-interacting protein of 47 kDa (TIP47), S3-12, and OXPAT. Members of this family are also present in evolutionarily distant organisms, including insects, slime molds and fungi. All PAT proteins share sequence similarity and the ability to bind intracellular lipid droplets, either constitutively or in response to metabolic stimuli, such as increased lipid flux into or out of lipid droplets. Positioned at the lipid droplet surface, PAT proteins manage access of other proteins (lipases) to the lipid esters within the lipid droplet core and can interact with cellular machinery important for lipid droplet biogenesis. Genetic variations in the gene for the best-characterized of the mammalian PAT proteins, perilipin, have been associated with metabolic phenotypes, including type 2 diabetes mellitus and obesity. In this review, we discuss how the PAT proteins regulate cellular lipid metabolism both in mammals and in model organisms