Regulation of apoptosis by viruses that infect insects.

Orthobunyviruses and alphaviruses cause encephalitis, neuronal apoptosis and mortality in mammals, but fail to kill the mosquitoes that transmit these viruses. Therefore, host cell factors, as well as viral factors, regulate the outcome of infection. Drosophila Reaper is a pro-death factor in the Drosophila nervous system during development but homologues were not previously known to exist outside flies. Recent discovery of a Reaper protein encoded by orthobunyaviruses provides interesting insight into the mechanisms by which viruses modulate the death pathway in both vertebrate and invertebrate hosts.

Insulin-mediated modifications of myocardial lipoprotein lipase and lipoprotein metabolism.

Recirculating organ perfusion in vitro was conducted with hearts from control rats, animals given a single dose of streptozotocin (65 mg/kg) 48 h earlier, and streptozotocin-treated rats administered insulin (5 units), 2 h prior to organ perfusion. During 45-min perfusions, the lipolysis of very low density lipoprotein (VLDL) triglyceride was significantly less in hearts from diabetics than in controls (41.9 +/- 7.3% of control). This was associated with significant reductions in heparin-releasable (functional) lipoprotein lipase and tissue lipoprotein lipase of perfused hearts. The decreases in VLDL triglyceride metabolism and the levels of myocardial lipoprotein lipase were completely reversed by treatment of diabetic rats with insulin 2 h prior to study. Similar improvement of VLDL triglyceride metabolism and increases in myocardial lipoprotein lipase activity were observed in hearts from diabetic rats by direct addition of 100 milliunits/ml of insulin to the recirculating perfusion media. Under these conditions, the increase in both fractions of lipoprotein lipase in response to insulin was completely inhibited, and utilization of VLDL triglyceride was partially inhibited by pre-perfusion with cycloheximide for 10 min. The data derived from either VLDL triglyceride lipolysis in organ perfusion or direct measurement of myocardial lipoprotein lipase demonstrate a direct effect of insulin on myocardial lipoprotein lipase activity, and suggest that the response to insulin may be due in part to effects on protein synthesis.

Metabolism of fatty acid, glycerol and a monoglyceride analogue by rat cardiac myocytes and perfused hearts.

Studies have been conducted on the uptake and metabolism of unesterified fatty acid, free glycerol and 1-hexadecyl glyceryl ether by rat cardiac myocytes, and of fatty acid, intact triglyceride and the glyceryl ether by perfused rat hearts. Cardiac myocytes efficiently extracted, oxidized and esterified oleic acid, but demonstrated little ability to utilize free glycerol. Although the glyceryl ether was efficiently extracted by myocytes, it was neither hydrolyzed or esterified. The perfused heart also extracted and metabolized unesterified fatty acid, and the fatty acid released during lipolysis of circulating lipoprotein triglyceride. The glyceride glycerol, however, was largely recovered (90%) in the perfusate suggesting inefficient myocardial utilization of either free glycerol or partial glycerides. Myocardial extraction of glyceryl monoether was demonstrated, but the monoglyceride analogue was also unmetabolized by intact heart tissue. The results suggest that if monoglycerides are produced by the action of lipoprotein lipase on circulating triglycerides, reutilization of intact monoglycerides for higher glyceride synthesis is not a major fate of these products.

Comparative metabolism of free and esterified fatty acids by the perfused rat heart and rat cardiac myocytes.

Studies have been conducted on the uptake and metabolism of unesterified oleic acid and lipoprotein triacylglycerol by the perfused rat heart, and of oleic acid, free glycerol and lipoprotein triacylglycerol by rat cardiac myocytes. The perfused heart efficiently extracted and metabolized unesterified fatty acid and the fatty acid released during lipolysis of the recirculating triacylglycerol. The released glyceride glycerol, however, was largely accumulated in the perfusion media. Cardiac myocytes also extracted and rapidly metabolized unesterified fatty acid. As with the intact heart, free glycerol was poorly utilized by cardiac myocytes. Although the cells appeared to extract a small amount of available extracellular triacylglycerol presented as very low density lipoprotein, this was shown to be unmetabolized, suggesting adsorption rather than surface lipolysis and uptake of the released fatty acid. The data suggest that myocytes are unable to metabolize triacylglycerol fatty acids without prior lipolysis by extracellular (capillary endothelial) lipoprotein lipase.

Lipoprotein lipase activity of adult rat cardiocytes.

Cardiocytes were prepared by enzymatic dissociation of adult rat ventricular tissue, and comparative studies on lipoprotein lipase activity were conducted on fresh homogenates and acetone powders of these cells. Lipolytic activity in fresh homogenates was largely dependent on addition of serum activator to the assay, and the activity was sensitive to 1 M NaCl. Lipoprotein lipase activity was maximized in acetone powder preparations of cardiocytes. Approx. 10% of the total lipolytic activity was extractable from acetone powders of cells homogenized in the absence of serum, while approx. 50% was soluble from powders of cells homogenized with 10% serum. The non-extractable lipolytic activity was inhibited 80% with 1 M NaCl and about 47% with antibodies (IgG) to heart lipoprotein lipase. The buffer-extracted enzyme was completely sensitive to NaCl and was inhibited 80% by low concentrations of anti-lipoprotein lipase antibodies.

Morphological and metabolic studies on adult cardiac myocytes.

We have previously described detailed procedures for isolating rat and canine myocytes from adult ventricular tissue, and have investigated ultrastructural and functional properties of these cells. The effects of isolation on cell morphology were tested by comparative scanning (SEM) and transmission electron microscopy (TEM) of adult canine cardiac myocytes. Cells cleanly separated at the lateral sarcolemmal borders and at the intercalated discs. Since the cells were contracted during fixation, SEM of the lateral cell borders showed longitudinal folds and transverse ridges. There were rows of regular protrusions corresponding to the mitochondria immediately beneath the sarcolemmal surface. Openings in the sarcolemmal membrane occurred at regular intervals at the level of the Z-bands and may represent the sites of externalization of the transverse tubular system. By SEM, the intercalated disc junction appeared as a microvillar border and this corresponded directly to the scalloped appearance seen by TEM. A controlled sonication procedure was developed to allow specific removal of the sarcolemma without disruption of underlying organelles. This allowed visualization of the cellular organization of myofibrils, mitochondria and the transverse tubular system. These cells retain biochemical and enzymatic characteristics of adult dog cardiac tissues such as ouabain and insulin sensitivity, fatty acid and lipoprotein uptake and metabolism, and prostaglandin synthesis. This cytological preparation permits the correlation of these types of metabolic studies with the known functions of isolated subcellular organelles of the myocardium.