Okadaic Acid, a Bioactive Fatty Acid fromHalichondria okadai, Stimulates Lipolysis in Rat Adipocytes: The Pivotal Role of Perilipin Translocation

Lipid metabolism in visceral fat cells is correlated with metabolic syndrome and cardiovascular diseases. Okadaic-acid, a 38-carbon fatty acid isolated from the black spongeHalichondria okadai, can stimulate lipolysis by promoting the phosphorylation of several proteins in adipocytes. However, the mechanism of okadaic acid-induced lipolysis and the effects of okadaic acid on lipid-droplet-associated proteins (perilipins and beta-actin) remain unclear. We isolated adipocytes from rat epididymal fat pads and treated them with isoproterenol and/or okadaic acid to estimate lipolysis by measuring glycerol release. Incubating adipocytes with okadaic acid stimulated time-dependent lipolysis. Lipid-droplet-associated perilipins and beta-actin were analyzed by immunoblotting and immunofluorescence, and the association of perilipin A and B was found to be decreased in response to isoproterenol or okadaic acid treatment. Moreover, okadaic-acid treatment could enhance isoproterenol-mediated lipolysis, whereas treatment of several inhibitors such as KT-5720 (PKA inhibitor), calphostin C (PKC inhibitor), or KT-5823 (PKG inhibitor) did not attenuate okadaic-acid-induced lipolysis. By contrast, vanadyl acetylacetonate (tyrosine phosphatase inhibitor) blocked okadaic-acid-dependent lipolysis. These results suggest that okadaic acid induces the phosphorylation and detachment of lipid-droplet-associated perilipin A and B from the lipid droplet surface and thereby leads to accelerated lipolysis.

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Tyrosine phosphorylation and cytoskeletal tension regulate the release of fibroblast adhesions.

The Journal of Cell Biology ◽

10.1083/jcb.131.2.525 ◽

1995 ◽

Vol 131 (2) ◽

pp. 525-537 ◽

Cited By ~ 93

Author(s):

E Crowley ◽

A F Horwitz

Keyword(s):

Tyrosine Phosphorylation ◽

Focal Adhesion ◽

Okadaic Acid ◽

Tyrosine Phosphatase ◽

Focal Adhesions ◽

Cell System ◽

Cell Contraction ◽

Permeabilized Cell ◽

Variable Intensity ◽

Associated Proteins

We have investigated the mechanisms by which fibroblasts release their adhesions to the extracellular matrix substrata using a permeabilized cell system in which the adhesions remain relatively stable. A large number of different molecules were assayed for their effect on focal adhesion stability using immunofluorescence with antibodies against different focal adhesion constituents. ATP uniquely stimulates a rapid breakdown of focal adhesions, and at high ATP concentrations (> 5 mM), many cells are released from the dish. The remaining cells appear contracted with talin, alpha-actinin, and vinculin localized diffusely throughout the cell. Integrin containing tracks of variable intensity outline the regions where cells had resided before they detached from the substratum. At lower ATP concentrations (0.5-5 mM) the cells remain spread; however the focal adhesion components, including integrin, show an array of phenotypes ranging from diffusely localized throughout the cell to a localization in small, thin focal adhesions. Okadaic acid, a serine, threonine phosphatase inhibitor, enhances the contracted phenotype, even at low concentrations (0.5 mM) of ATP. The localization of focal adhesion components is different in okadaic acid-treated cells. In highly contracted cells, integrin is present in tracks where the cells resided before the contraction; however focal adhesions are no longer apparent. Talin, vinculin, and alpha-actinin localize in trabecular networks toward the periphery of the cell. Interestingly, phosphotyrosine staining as well as nascent, intracellular integrin precedes the recruitment of focal adhesion constituents into the trabecular network. The ATP-stimulated focal adhesion breakdown appears to operate through two mechanisms. First, ATP stimulates the tyrosine phosphorylation of several cytoskeletally associated proteins. These tyrosine phosphorylations correlated well with focal adhesion breakdown. Furthermore, addition of a recombinant, constitutively active tyrosine phosphatase inhibits both the tyrosine phosphorylations and the breakdown of the focal adhesions. None of the major tyrosine phosphoproteins are FAK, integrin, tensin, paxillin, or other phosphoproteins implicated in focal adhesion assembly. The second mechanism is cell contraction. High ATP concentrations, or lower ATP concentrations in the presence of okadaic acid induce cell contraction. Inhibiting the contraction by addition of a heptapeptide IRICRKG, which blocks the actin-myosin interaction, also inhibits focal adhesion breakdown. Neither the peptide nor the phosphatase inhibits focal adhesion breakdown under all conditions suggesting that both tension and tyrosine phosphorylations mediate the release of adhesions.

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Subcellular localization of skeletal muscle lipid droplets and PLIN family proteins OXPAT and ADRP at rest and following contraction in rat soleus muscle

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00163.2011 ◽

2012 ◽

Vol 302 (1) ◽

pp. R29-R36 ◽

Cited By ~ 24

Author(s):

Rebecca E. K. MacPherson ◽

Eric A. F. Herbst ◽

Erica J. Reynolds ◽

Rene Vandenboom ◽

Brian D. Roy ◽

...

Keyword(s):

Skeletal Muscle ◽

Lipid Droplet ◽

Protein Interactions ◽

Lipid Droplets ◽

Droplet Surface ◽

Protein Protein Interactions ◽

Muscle Lipid ◽

Droplet Distribution ◽

Associated Proteins ◽

Skeletal Muscle Lipid

Skeletal muscle lipid droplet-associated proteins (PLINs) are thought to regulate lipolysis through protein-protein interactions on the lipid droplet surface. In adipocytes, PLIN2 [adipocyte differentiation-related protein (ADRP)] is found only on lipid droplets, while PLIN5 (OXPAT, expressed only in oxidative tissues) is found both on and off the lipid droplet and may be recruited to lipid droplet membranes when needed. Our purpose was to determine whether PLIN5 is recruited to lipid droplets with contraction and to investigate the myocellular location and colocalization of lipid droplets, PLIN2, and PLIN5. Rat solei were isolated, and following a 30-min equilibration period, they were assigned to one of two groups: 1) 30 min of resting incubation and 2) 30 min of stimulation ( n = 10 each). Immunofluorescence microscopy was used to determine subcellular content, distribution, and colocalization of lipid droplets, PLIN2, and PLIN5. There was a main effect for lower lipid and PLIN2 content in stimulated compared with rested muscles ( P < 0.05). Lipid droplet distribution declined exponentially from the sarcolemma to the fiber center in the rested muscles ( P = 0.001, r2= 0.99) and linearly in stimulated muscles (slope = −0.0023 ± 0.0006, P < 0.001, r2= 0.93). PLIN2 distribution declined exponentially from the sarcolemma to the fiber center in both rested and stimulated muscles ( P < 0.0001, r2= 0.99 rest; P = 0.0004, r2= 0.98 stimulated), while PLIN5 distribution declined linearly (slope = −0.0085 ± 0.0009, P < 0.0001, r2= 0.94 rest; slope=−0.0078 ± 0.0010, P = 0.0003, r2= 0.91 stimulated). PLIN5-lipid droplets colocalized at rest with no difference poststimulation ( P = 0.47; rest r2= 0.55 ± 0.02, stimulated r2= 0.58 ± 0.03). PLIN2-lipid droplets colocalized at rest with no difference poststimulation ( P = 0.48; rest r2= 0.66 ± 0.02, stimulated r2= 0.65 ± 0.02). Contrary to our hypothesis, these results show that PLIN5 is not recruited to lipid droplets with contraction in isolated skeletal muscle.

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Lipid Droplet-Associated Proteins Protect Renal Tubular Cells from Fatty Acid-Induced Apoptosis

American Journal Of Pathology ◽

10.2353/ajpath.2008.080137 ◽

2008 ◽

Vol 173 (5) ◽

pp. 1286-1294 ◽

Cited By ~ 19

Author(s):

Yoshimichi Urahama ◽

Yuki Ohsaki ◽

Yutaka Fujita ◽

Shoichi Maruyama ◽

Yukio Yuzawa ◽

...

Keyword(s):

Fatty Acid ◽

Lipid Droplet ◽

Tubular Cells ◽

Renal Tubular Cells ◽

Associated Proteins ◽

Renal Tubular ◽

Induced Apoptosis

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Skeletal muscle PLIN proteins, ATGL and CGI-58, interactions at rest and following stimulated contraction

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00418.2012 ◽

2013 ◽

Vol 304 (8) ◽

pp. R644-R650 ◽

Cited By ~ 50

Author(s):

Rebecca E. K. MacPherson ◽

Sofhia V. Ramos ◽

Rene Vandenboom ◽

Brian D. Roy ◽

Sandra J. Peters

Keyword(s):

Skeletal Muscle ◽

Lipid Droplet ◽

Protein Interactions ◽

Droplet Surface ◽

Gene Identification ◽

Protein Protein Interactions ◽

Muscle Lipid ◽

Associated Proteins ◽

Skeletal Muscle Lipid ◽

Intramuscular Lipid

Evidence indicates that skeletal muscle lipid droplet-associated proteins (PLINs) regulate lipolysis through protein-protein interactions on the lipid droplet surface. In adipocytes, PLIN1 is thought to regulate lipolysis by directly interacting with comparative gene identification-58 (CGI-58), an activator of adipose triglyceride lipase (ATGL). Upon lipolytic stimulation, PLIN1 is phosphorylated, releasing CGI-58 to fully activate ATGL and initiate triglyceride breakdown. The absence of PLIN1 in skeletal muscle leads us to believe that other PLIN family members undertake this role. Our purpose was to examine interactions between PLIN2, PLIN3, and PLIN5, with ATGL and its coactivator CGI-58 at rest and following contraction. Isolated rat solei were incubated for 30 min at rest or during 30 min of intermittent tetanic stimulation [150-ms volleys at 60 Hz with a train rate of 20 tetani/min (25°C)] to maximally stimulate intramuscular lipid breakdown. Results show that the interaction between ATGL and CGI-58 increased 128% following contraction ( P = 0.041). Further, ATGL interacts with PLIN2, PLIN3, and PLIN5 at rest and following contraction. The PLIN2-ATGL interaction decreased significantly by 21% following stimulation ( P = 0.013). Both PLIN3 and PLIN5 coprecipitated with CGI-58 at rest and following contraction, while there was no detectable interaction between PLIN2 and CGI-58 in either condition. Therefore, our findings indicate that in skeletal muscle, during contraction-induced muscle lipolysis, ATGL and CGI-58 strongly associate and that the PLIN proteins work together to regulate lipolysis, in part, by preventing ATGL and CGI-58 interactions at rest.

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The Lipid Droplet-Associated Protein Adipophilin Is Expressed in Human Trophoblasts and Is Regulated by Peroxisomal Proliferator-Activated Receptor-γ/Retinoid X Receptor

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/jc.2003-030628 ◽

2003 ◽

Vol 88 (12) ◽

pp. 6056-6062 ◽

Cited By ~ 55

Author(s):

Ibrahim Bildirici ◽

Cheong-Rae Roh ◽

W. Timothy Schaiff ◽

Beth M. Lewkowski ◽

D. Michael Nelson ◽

...

Keyword(s):

Fatty Acid ◽

Lipid Droplet ◽

Fatty Acid Uptake ◽

Cellular Fatty Acid ◽

Retinoid X Receptor ◽

Peroxisome Proliferator ◽

Acid Uptake ◽

Lipid Uptake ◽

Peroxisome Proliferator Activated Receptor ◽

Associated Proteins

Abstract Uptake and transplacental transfer of fatty acids is essential for fetal development. Human adipophilin and its murine ortholog adipocyte differentiation-related protein are lipid droplet-associated proteins that are implicated in cellular fatty acid uptake in adipocytes. The nuclear receptor peroxisome proliferator-activated receptor-γ (PPARγ) stimulates lipid uptake by adipocytes and enhances differentiation of placental trophoblasts. We therefore hypothesized that adipophilin is expressed in human trophoblasts and that its expression is regulated by PPARγ. We initially determined that adipophilin is expressed in human villous trophoblasts and that adipophilin expression is enhanced during differentiation of cultured primary term human trophoblasts. We also found that exposure of cultured human trophoblasts to the PPARγ ligand troglitazone resulted in a concentration-dependent increase in adipophilin expression. We observed a similar increase with LG268, a ligand for retinoid X receptor (RXR), the heterodimeric partner of PPARγ. Lastly, we demonstrated that ligand-activated PPARγ and RXR stimulated the transcriptional activity of adipophilin promoter in CV-1 cells and in the placental JEG3 cell line. We conclude that the expression of adipophilin is enhanced during trophoblast differentiation and is up-regulated by ligand-activated PPARγ/RXR. Enhanced adipophilin expression may contribute to fatty acid uptake by the placenta.

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