Cell surface changes during adipocyte differentiation in vitro.
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Biomedical subjects
Publications and source records attributed to A Cryer.
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When cardiac muscle cells from mature rats were incubated in vitro in the presence of heparin (8.7 nmole ml-1) lipoprotein lipase activity appeared in the incubation medium. The intracellular activity of the enzyme remained unchanged. Other glycosaminoglycans (heparan sulphate, dermatan sulphate, keratan sulphate and chrondroitin 6-sulphate) at the same or higher concentrations were totally ineffective in producing any enzyme redistribution between cells and medium. The release seen in the presence of heparin was blocked by the presence of cycloheximide. Cycloheximide by contrast had no effect on the release observed in the presence of dexamethasone, The action of endogenous glycosaminoglycans are unlikely therefore to have a significant role to play in the movement of lipoprotein lipase in heart tissue in vivo.
A comparison of the subfractions prepared from porcine plasma very low density lipoproteins by gel exclusion and heparin-Sepharose affinity chromatography revealed that the smallest and largest particles had the highest affinity for the glycosaminoglycan and had the highest ratio of apolipoprotein E to apolipoprotein CII. When the rates of triglyceride hydrolysis catalysed by lipoprotein lipase were compared for the subfractions the results were consistent with the view that apolipoprotein E may play a role in facilitating the catabolism of very low density lipoprotein triglyceride in the presence of glycosaminoglycan.
The lipoprotein composition and apoprotein composition of the major lipoprotein fraction (high density lipoprotein) were compared in White Carneau and Show Racer plasma. The capacity of the plasma and lipoproteins to activate the triacylglycerol hydrolyzing activity of lipoprotein lipase in vitro was compared in the two strains of birds and found to be identical in each case. It appears unlikely that differences in lipoprotein composition or tissue lipoprotein lipase activity will be reflected in the flux rates of lipoproteins in the two strains which have different susceptibilities to atherosclerosis.
Antisera with specific reactivity towards adipocyte cell surfaces were prepared and characterized. These preparations, absorbed to remove reactivities toward other cell types were used in an indirect labelled second antibody cellular immunoassay which distinguished bovine adipocyte precursors from fibroblasts and which allowed the progress of precursor cell differentiation in culture to be monitored with precision and sensitivity. The assay could be modified to use fluorescent, rather than radioactively-labelled, second antibody preparations and the changing reactivity of differentiating precursors to be visualized. Labelling of preparations in this way also allowed precursor cell populations to be analysed and quantitated using fluorescence-activated cell sorting technology.
Purified bovine milk lipoprotein lipase was shown to bind to intact porcine aortic endothelium in a specific, saturable fashion. The binding was reversed by exogenous heparin. A single class of binding sites was involved and at saturation 1.24 x 10(11) molecules of lipoprotein lipase/cm2 were bound. This represents 0.51 x 10(6) enzyme molecules per endothelial cell at a density of 1.2 x 10(3) molecules/micrometers 2. The enzyme binding was reduced by prior trypsinisation of the endothelial surface under conditions that removed cell surface glycosaminoglycan chains. The porcine endothelium was shown to have available at its surface 5.4 x 10(11) chains of heparan sulphate plus heparin-like glycosaminoglycans/cm2. Such an excess suggests that lipoprotein lipase may interact with approximately one in four of the available heparan sulphate chains.
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1. The lipoprotein lipase activity measured in acetone-ether powders of tissues from White Carneau and Show Racer pigeons was invariably somewhat lower in the former compared with the latter species. 2. At 100 and 200 Units of heparin per kg body weight the peak post-heparin lipolytic activity present in the plasma of White Carneau pigeons was significantly lower than that for Show Racers. At 50 Units per kg, this position was reversed. 3. It was concluded that the White Carneau pigeon may have an impaired functional lipoprotein lipase capacity compared to the Show Racer control.
Rainbow trout were exposed to cadmium in their aquarium water for different lengths of time and at different concentrations. More than 99% of the total body loads accumulated under all such conditions of exposure was found in the liver, kidney and gills. Comparison of the metal-binding proteins in these organs indicated that two low mol. wt proteins sequestered the cadmium while zinc was bound to metallothionein. Cadmium was not found in association with metallothionein unless artificially high concentrations were introduced either by intraperitoneal injection of the fish or by dialysis of tissue extracts in vitro.
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The types of collagen molecules synthesized by newly confluent rat and human adipocyte precursor cells were studied and compared with those synthesized by fibroblasts in culture. For this the cells were incubated in the presence of [2]3H]L-glycine and [5-3H]L-proline and the newly synthesized radiolabelled collagen produced was purified and analysed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. This analysis revealed that the ratio of Types I and III collagen produced by four adipocyte precursor cell strains, each of distinct origin, was identical to that found with two different strains of lung fibroblasts. The mean ratio of Type I: Type III collagen in all cases was approximately 3.5:1. Because mature fat cells do not synthesize collagen and the adipocyte precursor cells lose their capacity for collagen synthesis during their post-confluent differentiation in vitro these data provide a biochemical basis to substantiate previous suggestions, based on morphology only, that the adipocyte stem cell is fibroblastic in nature.
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Using an indirect, labelled-second-antibody cellular immunoassay technique, an adipocyte-specific antiserum has been investigated. Components of the antiserum were shown to bind to differentiated 3T3-L1 cells; the cellular capacity for binding increased progressively during the induced differentiation of these cells in vitro.
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