Synthesis and accumulation of a receptor regulatory protein associated with lipid droplet accumulation in 3T3-L1 cells.

Synthesis and accumulation of the recently identified prostaglandin F2alpha receptor regulatory protein (FPRP) was found to correlate closely with lipid droplet accumulation by 3T3-L1 preadipose cells. FPRP, a transmembrane glycoprotein, has been shown to regulate the binding of ligand to certain seven-transmembrane receptors. Anti-FPRP immunohistochemistry, Western blotting, and metabolic labeling/immunoprecipitation experiments demonstrated that FPRP was not detectable in undifferentiated 3T3-L1 cells. Interestingly, low levels of FPRP mRNA were detected in the undifferentiated 3T3-L1 cells. After induction of adipose differentiation, FPRP mRNA increased approximately 3 fold whereas FPRP synthesis increased approximately 50 fold. Differentiation induction with either dexamethasone/insulin/isobutylmethylxanthine or the thiazolidinedione derivative ADD 4743 were both effective at inducing FPRP accumulation and accumulation of lipid droplets. By co-immunohistochemical and lipid staining, greater than 99% of the cells accumulating lipid droplets possessed FPRP. FPRP mRNA and protein are also found in rat adipose tissue. Treatment of 3T3-L1 cells with an FPRP anti-sense oligonucleotide during differentiation decreased FPRP accumulation and resulted in a decrease in lipid droplets without altering the level of induction of a late marker of adipocyte differentiation, glycerol-3-phosphate dehydrogenase activity. Transient expression of an FPRP cDNA in undifferentiated 3T3-L1 cells was insufficient to induce lipid droplet accumulation.

Disruption of the vimentin intermediate filament system during adipose conversion of 3T3-L1 cells inhibits lipid droplet accumulation.

During the differentiation of 3T3-L1 pre-adipocytes, vimentin intermediate filaments are reorganized to form cage-like structures around the nascent lipid droplets. Initial studies with 3T3-L1 cells indicated that aggregation of vimentin filaments by nocodazole treatment during or shortly after induction of adipose conversion dramatically reduced the lipid droplet content of 3T3-L1 cells 96-120 hours after induction. Specific but transient disruption of vimentin following anti-IFA antibody injection also resulted in a decrease in lipid droplet formation in differentiating cells. To specifically and stably affect filament organization, 3T3-L1 cells lines were established by transfection with a glucocorticoid-regulatable, dominant negative mutant vimentin cDNA expression plasmid. Treatment of these cells (83 delta C) with dexamethasone resulted in expression of vimentin with a carboxyl-terminal deletion, which led to the disruption of the endogenous filament network. Induction of adipose conversion in 83 delta C cells lead to the formation of lipid droplets comparable to those seen in untransfected 3T3-L1 cells. Addition of dexamethasone during the adipose conversion of 83 delta C cells did not affect the induction of the marker enzyme glycerol-3-phosphate dehydrogenase or the incorporation of [14C]palmitate into triglycerides during a 10 minute pulse label. There was, however, a failure to form prominent lipid droplets and to accumulate [14C]palmitate-labeled triglycerides. Pulse-chase experiments indicated that the failure of these cells to accumulate triglyceride was associated with an increased rate of turnover. These studies indicate that vimentin filaments provide a function that influences lipid stability during adipose conversion of 3T3-L1 cells.

Variance cardiography for emergency department evaluation of chest pain patients.

OBJECTIVE: To determine the test performance of 24-lead variance cardiography (VC), an ECG technique that measures QRS morphologic variability, for ED evaluation of chest pain associated with coronary artery disease (CAD). METHODS: A prospective, single-blind study of VC was performed in a community teaching hospital ED. All chest pain patients (> 30 years of age) who, after initial emergency physician evaluation, were believed to have pain of potential cardiac etiology and were admitted to the hospital were eligible. Exclusion criteria included obvious noncardiac etiology for discomfort, bundle-branch block, atrial fibrillation, and incomplete subsequent cardiac evaluation. After initial evaluation and stabilization, VC was obtained. The numerical output of VC was a CAD index (CADI). Serum myoglobin and creatine kinase (CK)-MB levels were obtained at the time of presentation and after one, two, and six hours. Hospital records were reviewed to determine final diagnosis and in-hospital evaluation results. RESULTS: Fifty-two of 75 eligible patients had complete data. Final diagnoses were as follows: 27/52 (52%), noncardiac; 13/52 (25%), acute myocardial infarction (AMI); and 12/52 (23%), unstable angina due to CAD. Twenty-three percent (12/52) of the patients had CADIs < 75. Eleven of these were found to have noncardiac origins for their chest pain. The twelfth patient had a 12-lead ECG revealing AMI and had been given thrombolytic therapy with subsequent reperfusion prior to VC. Using a CADI < 75 as the cutoff for a negative study, VC alone had a negative predictive value of 92%, a sensitivity of 96%, a positive predictive value of 60%, and a specificity of 41%. CONCLUSION: A CADI < 75, in addition to clinical impression and initial ECG, may identify chest pain patients who do not have significant CAD. Further prospective assessment of VC is warranted.

The presence or absence of a vimentin-type intermediate filament network affects the shape of the nucleus in human SW-13 cells.

Human SW-13 cells express the intermediate filament protein vimentin in a mosaic pattern (Hedberg, K. K. and Chen, L. B. (1986). Exp. Cell Res. 163, 509-517). We have isolated SW-13 clones that do (vim+) or do not (vim-) synthesize vimentin as analyzed using anti-intermediate filament immunofluorescence, electron microscopy and two-dimensional gel analysis of detergent-extracted preparations. Vimentin is the only cytoplasmic intermediate filament protein present in the vim+ cells, and the vim- cells do not contain any detectable cytoplasmic intermediate filament system. The presence or absence of intermediate filaments did not observably affect the distribution of mitochondria, endoplasmic reticulum, microtubules or actin stress fibers when these structures were visualized by fluorescence microscopy. However, electron microscopy and anti-lamin A/C immunofluorescence studies showed that nuclear morphology in vim- cells was frequently characterized by large folds or invaginations, while vim+ cells had a more regular or smooth nuclear shape. When vim- cells were transfected with a mouse vimentin expression plasmid, the synthesis of a mouse vimentin filament network restored the smooth nuclear morphology characteristic of vim+ cells. Conversely, when vim+ cells were transfected with a carboxy-terminally truncated mutant vimentin, expression of the mutant protein disrupted the organization of the endogenous vimentin filaments and resulted in nuclei with a prominently invaginated morphology. These results indicated that in SW-13 cells the vimentin filament system affects the shape of the nucleus.