Cell Biology Symposium: imaging the organization and trafficking of lipolytic effectors in adipocytes

Abstract

The storage and mobilization of lipid energy are central functions of adipocytes. Lipid energy is stored as triglyceride in lipid droplet structures that are now recognized as bona fide organelles and whose functions are greatly influenced by members of the perilipin family of lipid droplet scaffolds. Recent work indicates that the signaling events underlying fatty acid mobilization involve protein trafficking to a specialized subset of lipid droplets. Furthermore, the core lipolytic machinery is composed of evolutionarily conserved proteins whose functions are conserved in avian and mammalian production species. Lipolysis affects many aspects of animal nutrition and physiology, which can have an important influence on growth efficiency, lactation, and meat quality. This review focuses on recent research that addresses the organization and trafficking of key players in hormone-stimulated lipolysis, and the central role of perilipin1A in adipocyte lipolysis. The review emphasizes recent work from the laboratories of the authors that utilizes imaging techniques to explore the organization and interactions among lipolytic effectors in live cells during lipolytic activation. A mechanistic understanding of lipolysis may lead to new strategies for promoting human and animal health.

Figure 1

Rapid regulation of adipocyte lipolysis of protein kinase A (PKA). Adipocyte lipolysis is regulated by diverse stimulatory (Stim.) and inhibitory (Inhib) ligands acting through G-protein coupled receptors (GPCR) that transverse the plasma membrane bilayer. G-protein coupled receptor-generated signals are integrated by adenylyl cyclase, which generates cAMP. In addition, insulin suppresses lipolysis through protein kinase B (PKB)-dependent activation of phosphodiesterase 3 (PDE3), which degrades cyclic adenosine monophosphate (cAMP) to adenosine monophosphate (AMP). Signals controlling lipolysis converge at PKA, which directly phosphorylates hormone-sensitive lipase (HSL) and perilipin1A (PLIN1A), the major PKA substrate in fat cells. Perilipin1A is targeted to the surface of lipid droplets that are covered by a phospholipid monolayer. Adipose triglyceride lipase (ATGL) and its co-activator, α/β hydrolase domain-containing 5 (ABHD5), are key elements PKA-regulated lipolysis, yet neither appears to be a direct target of PKA. Gs and Gi are the stimulatory and inhibitory G-proteins, respectively, of adenylyl cyclase.

Figure 2

Protein trafficking during initiation of lipolysis. Under basal conditions, perilipin1A (PLIN1A) and α/β hydrolase domain-containing 5 (ABHD5) form a complex on the surface of lipid droplets (LD). Hormone-sensitive lipase (HSL) is mainly cytosolic, whereas adipose triglyceride lipase (ATGL) is localized to LD, including those containing perilipin (PLIN). Stimulation by protein kinase A (PKA) activation leads to trafficking of HSL and ABHD5 (arrows). Phosphorylation of PLIN1A induces a conformational change that decreases its proximity to ABHD5, which allows ABHD5 to activate ATGL. Because ATGL acts exclusively on triglyceride (TG), it is likely that ATGL initiates generation of FFA. Phosphorylation of HSL promotes its translocation and tight association with PLIN1. A major component of HSL activity likely depends on generation of diglyceride (DG) substrate from the action of ATGL, whereas monoglyceride lipase (MG) acts to liberate glycerol and the final FFA. Not illustrated are potential effects of PLIN phosphorylation on the biophysical properties of the LD surface and the effects of sustained activity on LD fragmentation and movement.

Figure 3

Imaging protein trafficking in live cells by confocal fluorescence microscopy. Top row: Imaging the interaction of α/β hydrolase domain-containing 5 (ABHD5) and perilipin1A (PLIN1A) by fluorescence resonance energy transfer (FRET). In the basal state, ABHD5 and PLIN1A are in close molecular proximity and exhibit strong fluorescence energy transfer between enhanced cyan fluorescent protein (ECFP) and enhanced yellow fluorescent protein (EYFP) tags. Stimulation with forskolin rapidly decreases the association and is indicated by diminished FRET signal. Perilipin1A fused to EYFP shows the intracellular location of lipid droplets (LD). Middle row: Imaging the interaction of ABHD5 and adipose triglyceride lipase (ATGL) using bimolecular fluorescence complementation (BiFC). In the basal state, there is low interaction between ABHD5 and ATGL as indicated by weak fluorescence. Stimulation greatly increases the interaction, resulting in stronger BiFC signal, which co-localizes precisely to LD marked with PLIN1A-EYFP. Lower panel: Stimulationinduced accumulation of HSL on LD containing PLIN1A. In the basal state, ECFP-hormone sensitive lipase (HSL) is largely cytosolic, and stimulation leads to rapid and precise accumulation on LD-containing PLIN1A-EYFP.