Biomedical subjects

Anne Garcia

Publications and source records attributed to Anne Garcia.

6 recordsLinked to original sources

PubMed2006

The phosphorylation of serine 492 of perilipin a directs lipid droplet fragmentation and dispersion.

Perilipin A is a key regulator of triacylglycerol storage and hydrolysis in adipocytes; phosphorylation of perilipin A by protein kinase A facilitates maximal lipolysis. Chronic stimulation of lipolysis in 3T3-L1 adipocytes causes large perinuclear lipid droplets to fragment into myriad dispersed perilipin A-covered microlipid droplets. In cultured fibroblasts stably expressing ectopic perilipin A, clustered lipid droplets disperse throughout the cytoplasm upon incubation of the cells with forskolin and isobutylmethylxanthine (IBMX) to elevate levels of cAMP and activate protein kinase A, mirroring events observed in adipocytes. Furthermore, diethylum-belliferyl phosphate inhibits stimulated lipolysis but not the dispersion of lipid droplets, suggesting that products of lipolysis are not required for this remodeling process. We hypothesized that protein kinase A-mediated phosphorylation of perilipin A triggers the remodeling of lipid droplets. The mutation of serine 492 of perilipin A to alanine prevented the dispersion of clustered lipid droplets in fibroblasts stably expressing the mutated perilipin upon incubation with forskolin and IBMX. In contrast, the substitution of serines 81, 222, 276, or 433 with alanine, either singly or in combinations, did not affect the protein kinase A-mediated remodeling of lipid droplets. Interestingly, substitution of serines 433, 492, and 517 of perilipin A with glutamic acid residues blocked the dispersion of clustered lipid droplets in cells incubated with forskolin and IBMX, indicating that the addition of a negative charge does not mimic a phosphate group. We conclude that protein kinase A-mediated phosphorylation of serine 492 of perilipin A drives the fragmentation and dispersion of lipid droplets.

1-Methyl-3-isobutylxanthine↗

PubMed2006

Multi-detector row cardiac computed tomography accurately quantifies right and left ventricular size and function compared with cardiac magnetic resonance.

BACKGROUND: Cardiac magnetic resonance (CMR) accurately quantifies right ventricular (RV) and left ventricular (LV) volumes and function. Limited availability of CMR and increasing use of MR-incompatible cardiovascular devices underscore the potential utility of cardiac computed tomography (CT) for ventricular quantification. This study quantified biventricular size and systolic function with multi-detector row CT compared with CMR imaging. METHODS: Twenty-six subjects prospectively underwent CT and CMR examinations on a 16-detector CT and 1.5 T MR scanner, respectively; claustrophobia in one and nongated CT imaging in another precluded complete imaging in 2 subjects. Contiguous multiphase short-axis images were generated from axial CT data, and steady-state free precession cine MR produced contiguous short-axis cines. Semiautomated software generated ventricular borders to calculate volume, mass, and ejection fraction (EF) from both sets of images. Blinded observers completed quantification and wall motion analyses of 23 CMR and CT data sets independently. RESULTS: All measures of LV size and function by cardiac CT correlated well with CMR over a wide range of LV function (LVEF 30%-72% by CMR), including end-diastolic volume (r = 0.97), end-systolic volume (r = 0.97), EF (r = 0.97), and mass (r = 0.95). Of 24 cases, 6 had inadequate contrast opacification of the RV precluding RV segmentation. In the remaining 18 CMR-CT data pairs, RVEF showed moderate agreement (r = 0.86), and RV volumes correlated well (r = 0.97 and 0.94 for RV end-diastolic volume and RV end-systolic volume, respectively). Ten percent of LV segments visualized by CT were inadequate for wall motion assessment due to motion artifact or inadequate contrast between myocardium and endocardium. For segments adequately visualized by both techniques, the mean kappa statistic was 0.88 (range 0.78-1.0), consistent with good agreement. CONCLUSION: Cardiac CT accurately quantifies LV size and function; RV quantification with cardiac CT requires optimized contrast opacification of the RV.

Aged↗

PubMed2004

Hydrophobic sequences target and anchor perilipin A to lipid droplets.

Perilipins regulate triacylglycerol storage and hydrolysis in adipocytes. The central 25% of the perilipin A sequence, including three hydrophobic sequences (H1, H2, and H3) and an acidic region, targets and anchors perilipins to lipid droplets. Thus, we hypothesized that H1, H2, and H3 are targeting and anchoring motifs. We now show that deletion of any single hydrophobic sequence or combinations of H1 and H3 or H2 and H3 does not prevent targeting of the mutated perilipin to lipid droplets. In contrast, mutated perilipin lacking H1 and H2 showed reduced targeting, whereas perilipin lacking H1, H2, and H3 targeted poorly to lipid droplets; thus, H3 is a weak targeting signal and either H1 or H2 is required for optimal targeting. Complete elimination of perilipin targeting was observed only when all three hydrophobic sequences were deleted in combination with either the acidic region or N-terminal sequences predicted to form amphipathic beta-strands. Unlike intact perilipin A, mutated perilipin lacking either H1 and H2 or H1, H2, and H3 was released from lipid droplets after alkaline carbonate treatment, suggesting that these forms are loosely associated with lipid droplets. The three hydrophobic sequences play a major role in targeting and anchoring perilipins to lipid droplets.

3T3-L1 Cells↗

PubMed2004

Perilipin A mediates the reversible binding of CGI-58 to lipid droplets in 3T3-L1 adipocytes.

Perilipins, the major structural proteins coating the surfaces of mature lipid droplets of adipocytes, play an important role in the regulation of triacylglycerol storage and hydrolysis. We have used proteomic analysis to identify CGI-58, a member of the alpha/beta-hydrolase fold family of enzymes, as a component of lipid droplets of 3T3-L1 adipocytes. CGI-58 mRNA is highly expressed in adipose tissue and testes, tissues that also express perilipins, and at lower levels in liver, skin, kidney, and heart. Both endogenous CGI-58 and an ectopic CGI-58-GFP chimera show diffuse cytoplasmic localization in 3T3-L1 preadipocytes, but localize almost exclusively to the surfaces of lipid droplets in differentiated 3T3-L1 adipocytes. The localization of endogenous CGI-58 was investigated in 3T3-L1 cells stably expressing mutated forms of perilipin using microscopy. CGI-58 binds to lipid droplets coated with perilipin A or mutated forms of perilipin with an intact C-terminal sequence from amino acid 382 to 429, but not to lipid droplets coated with perilipin B or mutated perilipin A lacking this sequence. Immunoprecipitation studies confirmed these findings, but also showed co-precipitation of perilipin B and CGI-58. Remarkably, activation of cAMP-dependent protein kinase by the incubation of 3T3-L1 adipocytes with isoproterenol and isobutylmethylxanthine disperses CGI-58 from the surfaces of lipid droplets to a cytoplasmic distribution. This shift in subcellular localization can be reversed by the addition of propanolol to the culture medium. Thus, CGI-58 binds to perilipin A-coated lipid droplets in a manner that is dependent upon the metabolic status of the adipocyte and the activity of cAMP-dependent protein kinase.

1-Acylglycerol-3-Phosphate O-Acyltransferase↗

PubMed2003

The amino and carboxyl termini of perilipin a facilitate the storage of triacylglycerols.

Perilipin A is the most abundant lipid droplet-associated protein in adipocytes and serves important functions in regulating triacylglycerol levels by reducing rates of basal lipolysis and facilitating hormonally stimulated lipolysis. We have previously shown that the central region of perilipin A targets and anchors it to lipid droplets, at least in part via three moderately hydrophobic sequences that embed the protein into the hydrophobic core of the droplet. The current study examines the roles of the amino and carboxyl termini of perilipin A in facilitating triacylglycerol storage. Amino- and carboxyl-terminal truncation mutations of mouse perilipin A were stably expressed in 3T3-L1 preadipocytes, which lack perilipins. Triacylglycerol content of the cells was quantified as a measure of perilipin function and was compared with that of cells expressing full-length perilipin A or control cells lacking perilipins. The amino-terminal sequence between amino acids 122 and 222, including four 10-11-amino acid sequences predicted to form amphipathic beta-strands and a consensus site for cAMP-dependent protein kinase, and the carboxyl terminus of 112 amino acids that is unique to perilipin A were critical to facilitate triacylglycerol storage. The precocious expression of full-length perilipin A in 3T3-L1 preadipocytes aided more rapid storage of triacylglycerol during adipose differentiation. By contrast, the expression of highly truncated amino- or carboxyl-terminal mutations of perilipin failed to serve a dominant negative function in lowering triacylglycerol storage during adipose differentiation. We conclude that the amino and carboxyl termini are critical to the function of perilipin A in facilitating triacylglycerol storage.

3T3 Cells↗

PubMed2002

The central domain is required to target and anchor perilipin A to lipid droplets.

The perilipins are the most abundant proteins coating the surfaces of lipid droplets in adipocytes and are found at lower levels surrounding lipid droplets in steroidogenic cells. Perilipins drive triacylglycerol storage in adipocytes by regulating the rate of basal lipolysis and are also required to maximize hormonally stimulated lipolysis. To map the domains that target and anchor perilipin A to lipid droplets, we stably expressed fragments of perilipin A in 3T3-L1 fibroblasts. Immunofluorescence microscopy and immunoblotting of proteins from isolated lipid droplets revealed that neither the amino nor the carboxyl terminus is required to target perilipin A to lipid droplets; however, there are multiple, partially redundant targeting signals within a central domain including 25% of the primary amino acid sequence. A peptide composed of the central domain of perilipin A directed a fused green fluorescent protein to the surfaces of lipid droplets. Full-length perilipin A associates with lipid droplets via hydrophobic interactions, as shown by the persistence of perilipins on lipid droplets after centrifugation through an alkaline carbonate solution. Results of the mutagenesis studies indicate that the sequences responsible for anchoring perilipin A to lipid droplets are most likely domains of moderately hydrophobic amino acids located within the central 25% of the protein. Thus, we conclude that the central 25% of the perilipin A sequence contains all of the amino acids necessary to target and anchor the protein to lipid droplets.

Adipocytes↗