Biomedical subjects

Constantine Londos

Publications and source records attributed to Constantine Londos.

15 recordsLinked to original sources

PubMed2006

Functional compensation for adipose differentiation-related protein (ADFP) by Tip47 in an ADFP null embryonic cell line.

Ectopic accumulation of lipid droplets in non-adipose tissues correlates with the degree of insulin resistance in these tissues. Emerging evidence indicates that lipid droplets are specialized organelles that participate in lipid metabolism and intracellular trafficking. These properties are thought to derive from the lipid droplet-associated PAT protein family (perilipin, ADFP, and Tip47). The functions of the ubiquitously distributed adipose differentiation-related protein (ADFP) and Tip47 remain unknown. To evaluate the roles of ADFP and Tip47 in lipid biogenesis and metabolism, ADFP null and wild type (wt) clonal cell lines were established from ADFP null and wt mice, respectively. In ADFP null cells, Tip47 was identified as the sole lipid droplet-associated protein from the PAT family by mass spectroscopy, which was further confirmed by immunoblotting and immunocytochemistry. Following incubation with oleic acid, ADFP null cells were able to form lipid droplets to the same extent as wt cells. No statistical differences between the two cell types were observed in NEFA uptake or lipolysis. Small interference RNAs (siRNAs) against Tip47 were found to down-regulate protein levels for Tip47 by 85%. ADFP null cells treated with Tip47 siRNA retained the ability to form lipid droplets but to a lesser extent and shunted the utilization of exogenously added NEFA from triglycerides to phospholipids. These data support the hypothesis that Tip47 plays an important role in lipid metabolism. Tip47 and ADFP in peripheral tissues may play a critical role in regulating the formation and turnover, and hence metabolic consequences, of ectopic fat.

Adipocytes↗

PubMed2006

Role of adipose differentiation-related protein in lung surfactant production: a reassessment.

Based on data developed with the use of isolated lipid droplets from neonatal rat lung lipofibroblasts, we speculated previously that the droplet coat protein, adipose differentiation-related protein (ADFP), mediated the transfer of lipids into type 2 lung epithelial cells for the production of surfactant phospholipids. The present studies were designed to test the role of ADFP in this transfer with the use of ADFP-coated lipid droplets from CHO fibroblast cells and a cultured human lung epithelial cell line. We found no role for ADFP in the lipid transfer and conclude that a lipase associated with the lipid droplets hydrolyzes their core triacylglycerols, releasing fatty acids that are taken up by the epithelial cells.

Animals↗

PubMed2006

Decrease in intramuscular lipid droplets and translocation of HSL in response to muscle contraction and epinephrine.

A better understanding of skeletal muscle lipid metabolism is needed to identify the molecular mechanisms relating intramuscular triglyceride (IMTG) to muscle metabolism and insulin sensitivity. An increasing number of proteins have been reported to be associated with intracellular triglyceride (TG), among them the PAT family members: perilipin, ADRP (for adipocyte differentiation-related protein), and TIP47 (for tail-interacting protein of 47 kDa). Hormone-sensitive lipase (HSL) is thought to be the major enzyme responsible for IMTG hydrolysis in skeletal muscle. In adipocytes, regulation of HSL by intracellular redistribution has been demonstrated. The existence of such regulatory mechanisms in skeletal muscle has long been hypothesized but has never been demonstrated. The aim of this study was to characterize the PAT family proteins associated with IMTG and to investigate the effect of epinephrine stimulation or muscle contraction on skeletal muscle TG content and HSL intracellular distribution. Rat soleus muscles were either incubated with epinephrine or electrically stimulated for 15 min. Single muscle fibers were used for morphological analysis by confocal and transmission electron microscopy. We show a decrease in IMTG in response to both lipolytic stimuli. Furthermore, we identify two PAT family proteins, ADRP and TIP47, associated with IMTG. Finally, we demonstrate HSL translocation to IMTG and ADRP after stimulation with epinephrine or contraction.

Animals↗

PubMed2006

Optimized conditions for measuring lipolysis in murine primary adipocytes.

The current literature on lipolysis in murine primary adipocytes is rife with experiments performed under conditions not optimized for reproducible and reliable results. Here, we present conditions for optimizing the measurement of lipolysis in murine adipocytes. We demonstrate that adenosine management is of paramount importance in evaluating the lipolytic response under basal and stimulated conditions. Also, adipocyte concentrations in the 10,000-15,000 cells per milliliter range produce a greater increase in stimulated lipolysis than higher concentrations, and the response is further enhanced by agitating the cells.

Adenosine↗

PubMed2006

Degradation of perilipin is mediated through ubiquitination-proteasome pathway.

Perilipin protein coats the surface of intracellular lipid droplets and plays fundamental roles in lipid droplet formation and triacylglycerol hydrolysis. Perilipin is transcriptionally regulated through peroxisome proliferator-activated receptor and post-translationally stabilized by stored intracellular neutral lipids. In this study, we show that perilipin protein accumulates in transfected Chinese hamster ovary cells cultured in the presence of fatty acids but in turn is destabilized when lipid precursors for triacylglycerol synthesis are removed from culture serum. Adding fatty acids in the culture medium prevents the degradation of perilipin. Moreover, specific proteasome inhibitors, MG132, lactacystin, and ALLN, block the degradation, whereas inhibitors of other proteases are ineffective. Pulse-chase experiments confirm that perilipin is degraded through proteasome, a process that is inhibited by MG132 or ALLN and blunted by the addition of oleic acid. We have detected the co-immunoprecipitation of perilipin and ubiquitin, thus confirming that perilipin is conjugated to poly-ubiquitin and targeted for proteasomal degradation. Treatment with MG132 increases the expression of perilipin associated with lipid droplets as well as modestly throughout the cytosol. We conclude that the degradation of perilipin is mediated through an ubiquitination-proteasome pathway, which suggests another mode for the post-translational regulation of perilipin.

Animals↗

PubMed2005

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.

3T3-L1 Cells↗

PubMed2005

Effects of the human immunodeficiency virus-protease inhibitor, ritonavir, on basal and catecholamine-stimulated lipolysis.

Several of the aspartic acid protease inhibitors used to treat HIV infection increase basal lipolysis in adipocytes, but the cellular mechanisms leading to this augmentation are not well understood. We therefore studied the effects of chronic exposure to the HIV protease inhibitor, ritonavir, on the lipolytic cascade in 3T3-L1 adipocytes. Treatment of 3T3-L1 adipocytes with ritonavir for 14 d (during and after differentiation) enhanced basal, isoproterenol (Iso)-stimulated, and cAMP analog-stimulated lipolysis. Enhancement of lipolysis was observed after Iso at concentrations between 0.1 and 10 mum. Despite a significant decrease in cyclic nucleotide phosphodiesterase (PDE)3B activity and protein levels, there were no changes in Iso-stimulated intracellular cAMP, protein kinase A (PKA) expression, or PKA activity. Ritonavir-augmented lipolysis was also observed under conditions that reversed the effect on PDE3B activity via preincubation with 1 mum (-)-N(6)-(2-phenylisopropyl)adenosine. In ritonavir-treated cells, protein expression of the lipid droplet-protective protein, perilipin, was significantly decreased, whereas there was no change in hormone-sensitive lipase. Activation of ERK1/2 by Iso did not play a role in the augmentation. We conclude that ritonavir decreases PDE3B and perilipin protein expression and affects both basal and catecholamine-stimulated lipolysis in 3T3-L1 adipocytes primarily through actions at sites downstream of PKA.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed2004

Physical association between the adipocyte fatty acid-binding protein and hormone-sensitive lipase: a fluorescence resonance energy transfer analysis.

Previous in vitro studies have established that hormone sensitive lipase (HSL) and adipocyte fatty acid-binding protein (AFABP) form a physical complex that presumably positions the FABP to accept a product fatty acid generated during catalysis. To assess AFABP-HSL interaction within a cellular context, we have used lipocytes derived from 293 cells (C8PA cells) and examined physical association using fluorescence resonance energy transfer. Transfection of C8PA cells with cyan fluorescent protein (CFP)-HSL, yellow fluorescent protein (YFP)-adipocyte FABP, or YFP-liver FABP revealed that under basal conditions each protein was cytoplasmic. In the presence of 20 microm forskolin, CFP-HSL translocated to the triacylglycerol droplet, coincident with BODIPY-FA labeled depots. Fluorescence resonance energy transfer analysis demonstrated that CFP-HSL associated with YFP-adipocyte FABP in both basal and forskolin-treated cells. In contrast, little if any fluorescence resonance energy transfer could be detected between CFP-HSL and YFP-liver FABP. These results suggest that a pre-lipolysis complex containing at least AFABP and HSL exists and that the complex translocates to the surface of the lipid droplet.

Adipocytes↗

PubMed2004

Structure of a lipid droplet protein; the PAT family member TIP47.

The perilipin/ADRP/TIP47 (PAT) proteins localize to the surface of intracellular neutral lipid droplets. Perilipin is essential for lipid storage and hormone regulated lipolysis in adipocytes, and perilipin null mice exhibit a dramatic reduction in adipocyte lipid stores. A significant fraction of the approximately 200 amino acid N-terminal region of the PAT proteins consists of 11-mer helical repeats that are also found in apolipoproteins and other lipid-associated proteins. The C-terminal 60% of TIP47, a representative PAT protein, comprises a monomeric and independently folded unit. The crystal structure of the C-terminal portion of TIP47 was determined and refined at 2.8 A resolution. The structure consists of an alpha/beta domain of novel topology and a four-helix bundle resembling the LDL receptor binding domain of apolipoprotein E. The structure suggests an analogy between PAT proteins and apolipoproteins in which helical repeats interact with lipid while the ordered C-terminal region is involved in protein:protein interactions.

Amino Acid Sequence↗

PubMed2004

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.

Adipocytes↗

PubMed2003

Targeting of big stanniocalcin and its receptor to lipid storage droplets of ovarian steroidogenic cells.

Stanniocalcin (STC) is a large polypeptide hormone that is widely distributed in tissues such as kidney, adrenal, and ovary. In most tissues, STC exists as a 50-kDa homodimer (STC50). The ovaries produce a higher molecular weight variant (big STC) in androgen-producing theca cell and interstitial cell compartments. Luteal cells, which do not express the STC gene, nonetheless contain high levels of STC protein, suggesting they are targeted by and sequester big STC through a receptor-mediated process. Recently, an STC.alkaline phosphatase fusion protein was used to characterize mitochondrial targeting and sequestration of STC50 and its receptor in liver and kidney. The main objective of the present study was to characterize big STC and its receptor in mammalian ovary and determine whether the ovarian STC variant was similarly targeted to luteal cell mitochondria. By in situ ligand binding, we identified large numbers of STC receptors on corpus luteal cells. However, a more detailed analysis of sub-cellular fractions revealed that both STC and its receptor were not preferentially targeted to mitochondria but instead to cholesterol/lipid storage droplets, which was more indicative of a role in steroidogenesis. Functional studies revealed that additions of big STC had concentration-dependent inhibitory effects on both basal and stimulated progesterone output by primary cultured luteal cells. Furthermore, STC receptor levels were up-regulated in luteal cells in response to protein kinase A activation. Taken together, these findings indicate that theca cell-derived big STC is targeted to the cholesterol/lipid storage droplets of luteal cells to regulate steroidogenesis. This constitutes the first reported description of polypeptide hormone and receptor targeting to cholesterol/lipid droplets and the first biological role for the big STC variant.

Animals↗

PubMed2003

Mutational analysis of the hormone-sensitive lipase translocation reaction in adipocytes.

Lipolysis in adipocytes governs the release of fatty acids for the supply of energy to various tissues of the body. This reaction is mediated by hormone-sensitive lipase (HSL), a cytosolic enzyme, and perilipin, which coats the lipid droplet surface in adipocytes. Both HSL and perilipin are substrates for polyphosphorylation by protein kinase A (PKA), and phosphorylation of perilipin is required to induce HSL to translocate from the cytosol to the surface of the lipid droplet, a critical step in the lipolytic reaction (Sztalryd C., Xu, G., Dorward, H., Tansey, J. T., Contreras, J.A, Kimmel, A. R., and Londos, C. (2003) J. Cell Biol. 161, 1093-1103). In the present paper we demonstrate that phosphorylation at one of the two more recently discovered PKA sites within HSL, serines 659 and 660, is also required to effect the translocation reaction. Translocation does not occur when these serines residues are mutated simultaneously to alanines. Also, mutation of the catalytic Ser-423 eliminates HSL translocation, showing that the inactive enzyme does not migrate to the lipid droplet upon PKA activation. Thus, HSL translocation requires the phosphorylation of both HSL and perilipin.

3T3-L1 Cells↗

PubMed2003

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 beta-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.

Adipocytes↗

PubMed2002

Functional studies on native and mutated forms of perilipins. A role in protein kinase A-mediated lipolysis of triacylglycerols.

Perilipin A coats the lipid storage droplets in adipocytes and is polyphosphorylated by protein kinase A (PKA); the fact that PKA activates lipolysis in adipocytes suggests a role for perilipins in this process. To assess whether perilipins participate directly in PKA-mediated lipolysis, we have expressed constructs coding for native and mutated forms of the two major splice variants of the perilipin gene, perilipins A and B, in Chinese hamster ovary fibroblasts. Perilipins localize to lipid droplet surfaces and displace the adipose differentiation-related protein that normally coats the droplets in these cells. Perilipin A inhibits triacylglycerol hydrolysis by 87% when PKA is quiescent, but activation of PKA and phosphorylation of perilipin A engenders a 7-fold lipolytic activation. Mutation of PKA sites within the N-terminal region of perilipin abrogates the PKA-mediated lipolytic response. In contrast, perilipin B exerts only minimal protection against lipolysis and is unresponsive to PKA activation. Since Chinese hamster ovary cells contain no PKA-activated lipase, we conclude that the expression of perilipin A alone is sufficient to confer PKA-mediated lipolysis in these cells. Moreover, the data indicate that the unique C-terminal portion of perilipin A is responsible for its protection against lipolysis and that phosphorylation at the N-terminal PKA sites attenuates this protective effect.

Animals↗

PubMed2002

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 as Drosophila 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.

Adipose Tissue↗