A central function of adipocytes is the storage and mobilization of energy in the form of triglyceride. A considerable amount is known about the enzymatic basis for lipogenesis and lipolysis; however, our understanding of how these processes are organized and regulated within cells is incomplete. Until recently, cellular triglyceride was considered to be stored in droplets lacking biological structure or organization. Growing evidence, however, suggests that lipid droplets are specialized, heterogeneous organelles that perform distinct roles in lipid biosynthesis, transport, and mobilization (1-3). A large number of proteins have been found to change their associations with lipid droplets in response to lipolytic stimulation. How these proteins are coordinated to control lipid storage and utilization remains largely unknown.It is well established that activation of cAMP-dependent kinase (PKA) 3 is the major signaling mechanism by which hormones and neurotransmitters stimulate lipolysis in adipocytes (4, 5). Most work regarding hormone stimulated lipolysis has focused on perilipin (Plin), a protein that binds to the surface of certain lipid droplets (LDs), and hormone-sensitive lipase (HSL), which translocates to lipid during lipolytic activation.Plin is the major target for PKA-mediated phosphorylation in adipocytes (6, 7), appears to be essential for hormone-stimulated lipolysis (8) and exerts both positive and negative effects on lipolytic rate (9 -11). Plin was originally viewed as a physical barrier that passively regulated access of lipases to store triglyceride; however, more recent data indicates that Plin may function more as a LD scaffold that directs the trafficking of lipolytic proteins to a specialized subcellular domain (11). Nonetheless, the temporal and spatial relationships between Plin and other proteins in the lipolytic pathway during PKA activation remain ill-defined.PKA activation promotes the translocation of HSL to lipid, which correlates with the magnitude of stimulated lipolysis (7,12). Work from several laboratories indicates that Plin and HSL synergize to trigger PKA-mediated lipolysis (11, 13), and our recent work indicates that HSL specifically translocates to a subset of LDs containing Plin (11). It is presently unclear whether Plin and HSL interact directly, whether the interaction takes place on certain LDs and not others, or whether the interaction occurs on a timescale that is compatible with the initiation of lipolysis.The complexity of regulated lipolysis has recently expanded with the identification of adipose triglyceride lipase (Atgl) (14, * This work was supported in part by grants from the National Institutes of Health (DK 62292), the American Diabetes Association, the Fund for Medical Research and Education at WSU, and the Joseph Young Research Fund. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fac...

Selective agonists of beta(3)-adrenergic receptors (Adrb3) exhibit potent anti-diabetes properties in rodent models when given chronically, yet the mechanisms involved are poorly understood. A salient feature of chronic Adrb3 activation is pronounced remodeling of white adipose tissue (WAT), which includes mitochondrial biogenesis and elevation of metabolic rate. To gain insights into potential mechanisms underlying WAT remodeling, the time course of remodeling induced by the Adrb3 agonist CL-316,243 (CL) was analyzed using histological, physiological, and global gene profiling approaches. The results indicate that continuous CL treatment induced a transient proinflammatory response that was followed by cellular proliferation among stromal cells and multilocular adipocytes. CL treatment strongly fragmented the central lipid storage droplet of mature adipocytes and induced mitochondrial biogenesis within these cells. Mitochondrial biogenesis was correlated with the upregulation of genes involved in fatty acid oxidation and mitochondrial electron transport activity. The elevated catabolic activity of WAT was temporally correlated with upregulation of peroxisome proliferator-activated receptor-alpha and its target genes, suggesting involvement of this transcription factor in coordinating the gene program that elevates WAT catabolic activity.

Regulation of triglyceride storage and mobilization is critically dependent on the subcellular targeting and trafficking of specific proteins. Recent work demonstrates that this trafficking involves scaffold proteins of the perilipin (Plin) 2 family, including those that are ubiquitously expressed, such as Plin2 (adipose differentiation-related protein) and Plin3 (tail-interacting protein 47, TIP47), and those with restricted expression, such as Plin1 (perilipin) and Plin5 (muscle lipid droplet protein) that appear to have specialized functions (1). Although each Plin homolog has a conserved Plin domain (pfam 03036), amino acid sequences of family members diverge widely outside of this domain (2). Nonetheless, the sequences of individual orthologs are well conserved in mammals and suggest that important functions might be mediated by sequences outside of the Plin domain.We have been investigating how Plin family members organize and regulate the trafficking of lipolytic effector proteins and have focused on Plin1 and Plin5 (3-6). Plin1 is expressed almost exclusively in adipose tissues and plays a central role in the storage of triglyceride and in the rapid mobilization of fatty acids by activators of protein kinase A (1). Recent work indicates that one means by which Plin1 regulates triglyceride storage and mobilization is by controlling the availability of ␣-␤-hydrolase domain-containing 5 (Abhd5), a potent activator of adipose triglyceride lipase (Atgl) (4).In contrast, Plin5 is highly expressed in tissues that have high rates of fatty acid oxidation, such as heart, skeletal muscle, and liver (7,8). Interestingly, expression of Plin5 promotes both triglyceride storage and fatty acid oxidation. Plin5 expression is up-regulated by peroxisome proliferator-activated receptor ␣, and this regulation appears to be part of an expression program that shifts the metabolism of cells from fatty acid storage to oxidation (7).Lipolysis occurs on the surface of intracellular lipid droplets, and several lines of evidence indicate that droplet targeting is critical to the cellular function of Abhd5 and Atgl (9, 10). Abhd5 is targeted to lipid droplets via direct interactions with Plin1 and Plin5 (5, 10, 11). However, unlike Plin1, Plin5 expression promotes the colocalization and interaction of Abhd5 and Atgl in unstimulated cells, which facilitates lipolysis (3-5). It is not known how Plin5 coordinates the interaction of Atgl and Abhd5. On the one hand, Plin5 could bind Atgl5 directly. Alternatively, Atgl might be recruited to the Plin5-containing lipid droplets by virtue of its interaction with Abhd5.In the experiments below, we determined that Atgl interacts with Plin5 but not Plin1. Interestingly, although Plin5 binds both Abhd5 and Atgl, the same Plin5 molecule does not bind both at the same time. Protein complementation experiments, however, indicate that Plin5 forms homo-oligomers and suggest that Abhd5 and Atgl interact as part of this oligomeric structure. Analysis of chimeric and truncated Plin proteins demonstrates ...

Adrenergic receptors (AR) are nearly exclusively expressed in brown and white adipose tissues, and chronic activation of these receptors by selective agonists has profound anti-diabetes and anti-obesity effects. This study examined metabolic responses to acute and chronic ␤ 3-AR activation in wild-type C57Bl/6 mice and congenic mice lacking functional uncoupling protein (UCP)1, the molecular effector of brown adipose tissue (BAT) thermogenesis. Acute activation of ␤3-AR doubled metabolic rate in wild-type mice and sharply elevated body temperature and BAT blood flow, as determined by laser Doppler flowmetry. In contrast, ␤3-AR activation did not increase BAT blood flow in mice lacking UCP1 (UCP1 KO). Nonetheless, ␤ 3-AR activation significantly increased metabolic rate and body temperature in UCP1 KO mice, demonstrating the presence of UCP1-independent thermogenesis. Daily treatment with the ␤3-AR agonist CL-316243 (CL) for 6 days increased basal and CL-induced thermogenesis compared with naive mice. This expansion of basal and CL-induced metabolic rate did not require UCP1 expression. Chronic CL treatment of UCP1 KO mice increased basal and CL-stimulated metabolic rate of epididymal white adipose tissue (EWAT) fourfold but did not alter BAT thermogenesis. After chronic CL treatment, CLstimulated thermogenesis of EWAT equaled that of interscapular BAT per tissue mass. The elevation of EWAT metabolism was accompanied by mitochondrial biogenesis and the induction of genes involved in lipid oxidation. These observations indicate that chronic ␤3-AR activation induces metabolic adaptation in WAT that contributes to ␤ 3-ARmediated thermogenesis. This adaptation involves lipid oxidation in situ and does not require UCP1 expression.

Cellular lipid metabolism is regulated in part by protein-protein interactions near the surface of intracellular lipid droplets. This work investigated functional interactions between Abhd5, a protein activator of the lipase Atgl, and Mldp, a lipid droplet scaffold protein that is highly expressed in oxidative tissues. Abhd5 was highly targeted to individual lipid droplets containing Mldp in microdissected cardiac muscle fibers. Mldp bound Abhd5 in transfected fibroblasts and directed it to lipid droplets in proportion to Mldp concentration. Analysis of protein-protein interactions in situ demonstrated that the interaction of Abhd5 and Mldp occurs mainly, if not exclusively, on the surface of lipid droplets. Oleic acid treatment rapidly increased the interaction between Abhd5 and Mldp, and this effect was suppressed by pharmacological inhibition of triglyceride synthesis. The functional role of the Abhd5-Mldp interaction was explored using a mutant of mouse Abhd5 (E262K) that has greatly reduced binding to Mldp. Mldp promoted the subcellular colocalization and interaction of Atgl with wild type, but not mutant, Abhd5. This differential interaction was reflected in cellular assays of Atgl activity. In the absence of Mldp, wild type and mutant Abhd5 were equally effective in reducing lipid droplet formation. In contrast, mutant Abhd5 was unable to prevent lipid droplet accumulation in cells expressing Mldp despite considerable targeting of Atgl to lipid droplets containing Mldp. These results indicate that the interaction between Abhd5 and Mldp is dynamic and essential for regulating the activity of Atgl at lipid droplets containing Mldp.Growing evidence indicates that lipogenesis and lipolysis are regulated by protein-protein interactions that occur on the surface of specialized intracellular lipid droplets (1, 2). PAT 3 (perilipin, adipophilin, and TIP-47) proteins, are thought to be key regulators of these processes by serving as scaffolds that organize and regulate the protein trafficking at lipid droplet surfaces (1-3). Mldp (muscle lipid droplet protein; alternatively, OXPAT, LSDP5) is a PAT family member that is highly expressed in tissues, like muscle and liver, having high oxidative capacity (4 -6). Expression of Mldp is up-regulated under conditions such as fasting and diabetes, in which the systemic supply of lipid to target tissues is increased, and in vitro studies suggest that Mldp plays a role in facilitating triglyceride storage as well as fatty acid oxidation (4 -6). It is not presently known how Mldp is involved in these functions, but we hypothesize that it is likely to involve direct or indirect interactions with lipases and lipase co-activators (3, 7). Abhd5 (␣/␤ hydrolase domain-containing protein 5; alternatively CGI-58) is an evolutionarily conserved protein that acts as a potent activator of Atgl (adipose triglyceride lipase; alternatively, PNPLA2, desnutrin, TTS-2.1) (8). Both proteins are expressed in a variety of tissues, and rare homozygous mutations of either gene in humans produces a...