J.E. Steele

Hormonal stimulation of lipid transport in the American cockroach, Periplaneta americana

Abstract The corpus cardiacum-allatum complex of the cockroach, Periplaneta americana , contains a factor which causes decreased levels of triglycerides and diglycerides in hemolymph. Coincident with these changes are an increase in the concentration of the glycerides within the fat body. The evidence suggests that triglycerides are converted to diglycerides in the hemolymph prior to their transport into the fat body.

The preparation of trophocytes from disaggregated fat body of the cockroach (Periplaneta americana)

A method has been developed for disaggregating the fat body of the adult American cockroach, Periplaneta americana, using collagenase. The yield of cells is sensitive to the osmolarity of the dispersing medium and to the age of the cockroaches from which the fat bodies are taken. Trophocytes uncontaminated with other cells were obtained by taking advantage of the low density of these cells which causes them to float to the top of the dispersion medium. In contrast, the mycetocytes and urocytes being denser than the medium sink to the bottom. The trophocytes retain the ability to respond to the synthetic hyperglycaemic hormones, CCI and CCII, as shown by the activation of phosphorylase and the stimulation of trehalose efflux. The trophocytes incorporated leucine into protein secreted by the cells in a time dependent manner.

Hypertrehalosemic hormones increase the concentration of free fatty acids in trophocytes of the cockroach (Periplaneta americana) fat body

The effect of the hypertrehalosemic hormones, HTH-I and HTH-II, on free fatty acid levels in trophocytes prepared from Periplaneta americana fat body by collagenase treatment was investigated. Following a challenge from either of the hormones the content of palmitic, stearic, oleic, and linoleic acid in the trophopcytes increased. The increase in free fatty acid concentration due to the action of the synthetic hormones was generally in the range of 40–95% for each of the four fatty acids. Crude corpus cardiacum extract containing the native hormone also had a stimulatory effect, which was comparable to that of the synthetic hormones. In the intact insect the injection of synthetic hormone was followed by an increase in the level of the four fatty acids in the hemolymph. HTH-II was more potent in this respect than HTH-I. Free fatty acids in the mycetocytes and urate cells did not respond to either of the synthetic hormones.

Control of phospholipase A2 activity in cockroach (periplaneta americana) fat body trophocytes by hypertrehalosemic hormone: the role of calcium

Recently, synthetic HTH-I and HTH-II have been shown to increase the formation of free fatty acids in cockroach (Periplaneta americana) fat body. In this study we show that HTH-II increases PLA(2) activity in dispersed trophocytes, thus implying that phospholipid is a potential source of the fatty acids. The increase in HTH-induced PLA(2) activity is triggered by an increase in [Ca(2+)](i) but extracellular Ca(2+) is also required for a maximal Ca(2+) signal: an effect that can be blocked by the introduction of BAPTA into the trophocytes. Treating trophocytes with ryanodine blocks the increase in PLA(2) activity that follows treatment of the cells with HTH-II. This indicates that the Ca(2+) release channels are distinct from those that respond to inositol trisphosphate. Thapsigargin, which releases Ca(2+) to the cytosol from an intracellular store, increases PLA(2) activity. The data show that the enzyme is translocated from the cytosol to the plasma membrane.

Hormonal activation of phosphorylase in cockroach fat body trophocytes: A correlation with trans‐membrane calcium flux

This study is an investigation of the temporal relationship between transmembrane Ca(2+) fluxes, and glycogen phosphorylase activation in dispersed trophocytes from the fat body of the cockroach, Periplaneta americana. Phosphorylase is maximally activated within 5 min after treating the trophocytes with either of the hypertrehalosemic hormones, Pea-HTH-I and Pea-HTH-II. Activation caused by Pea-HTH-II is sustained for a longer period than that produced by Pea-HTH-I. Chelation of extracellular Ca(2+) with EGTA blocks the activation of phosphorylase by HTH. Similarly, chelation of intracellular Ca(2+) with Quin 2 greatly diminishes the phosphorylase activating effect of both HTHs. The data support the view that an increase in the intracellular Ca(2+ )concentration is required for the activation of phosphorylase and that extracellular Ca(2+) is an essential, although not necessarily sole, source of Ca(2+) for this purpose. Using (45)Ca(2+) to trace the movement of Ca(2+) following a challenge with either Pea-HTH-I or -II, it was shown that (45)Ca(2+)influx nearly doubled during the first 30 s. At this time, the trophocytes begin to expel Ca(2+) at a rate higher than that of untreated cells and this state persists for approximately 4 min. The Ca(2+) fluxes are consistent with its postulated role in the activation of phosphorylase. Arch.

A novel function of cockroach (Periplaneta americana) hypertrehalosemic hormone : translocation of lipid from hemolymph to fat body

The hypertrehalosemic hormones Pea HTH-I and Pea HTH-II have a lipogenic effect in the cockroach, Periplaneta americana. This has been determined by measuring the change in tri-, di-, monoacylglycerol, phospholipid and free fatty acids in the hemolymph and fat body in response to the hormones. The effect of both hormones 6h after injection is comparable and is expressed as an increase in fat body triacylglycerol and a decrease in hemolymph triacylglycerol. Coincident with this effect the concentration of diacylglycerol in the hemolymph decreases while that in the fat body increases. Hemolymph and fat body phospholipid are both lowered by HTH. The concentration of free fatty acids in the hemolymph, as well as fat body, are increased by both hormones. Confirmation of the lipogenic effect of HTH has been obtained by feeding cockroaches [3H]oleic acid and measuring the incorporation of label into hemolymph and fat body lipid. The data show that the specific activity of the hemolymph phospholipid fraction increases more than 50%. In fat body, the specific activity of the label in the triacylglycerol fraction increased more than 2-fold while that in the free fatty acid fraction increased more than 20-fold. The data support the view that the fatty acids incorporated into fat body triacylglycerol under the influence of HTH are not formed de novo but are derived from the sn-1 and sn-2 positions of phospholipid which is initially formed in the midgut wall.

Carbohydrate and lipid metabolism in cockroach (Periplaneta americana) fat body are both activated by low and similar concentrations of Peram-AKH II

Injection of 0.1 pmol of the octapeptide Peram-AKH II (pGlu-Leu-Thr-Phe-Thr-Pro-Asn-TrpNH(2)) elicits a significant hypertrehalosemic response in the American cockroach, Periplaneta americana; a maximal effect is obtained with 1pmol. The latter amount also lowers the level of neutral lipid (NL) and phospholipid (PL) in the hemolymph. The evidence supports the idea that Peram-AKH II promotes the liberation of fatty acids from hemolymph phospholipid, and indirectly diacylglycerol in the same compartment. The fatty acids are then transported into the fat body where they are converted into triacylglycerol for storge. Because lipolysis and trehalose synthesis are initiated by a common concentration of Peram-AKH II it is reasonable to suggest that the physiological function of Peram-AKH II involves the participation of both metabolic pathways.

Lipid metabolism in the cockroach, Periplaneta americana, is activated by the hypertrehalosemic peptide, HTH-I

Phosphatidylcholine and phosphatidylethanolamine are the major constituents of the phospholipid pool in cockroach (Periplaneta americana) fat body and hemolymph. Both species of phospholipid are significantly decreased 6h after injecting hypertrehalosemic hormone I (HTH-I) into the hemocoel. Loss of phospholipid is accompanied by an accumulation of the phospholipid degradation products glycerophosphorylcholine and glycerol. HTH-I also increases phospholipase activity in the hemolymph and this is thought to be responsible for the depletion of hemolymph phospholipid. Phospholipase activity peaks approximately 2h after injection of HTH-I and returns to normal at 6h. In vitro, total phospholipid in the fat body is decreased by HTH-I whereas the concentration of diacylglycerol displays a corresponding increase. HTH-I elevates free fatty acid levels but has no effect on triacylglycerol. These effects of HTH-I are blocked by the phospholipase inhibitor mepacrine.

Regulation of fat body mitochondrial respiration in Periplaneta americana by a novel factor from the corpus cardiacum

Respiration of fat body (Periplaneta americana) mitochondria is increased by pretreatment of the tissue with corpus cardiacum (CC) extract. The magnitude of the increase depends on the type of substrate supplied for oxidation. With 5 mM pyruvate the respiration increased 22%, decreasing to 0 with 1 mM pyruvate. In contrast, 50 microM and 0.2 mM palmitic acid supported an increase in CC-stimulated respiration of 14 and 44%, respectively. Unlike crude CC extract, the synthetic hyperglycemic peptides CCI and CCII failed to alter the respiratory activity of fat body mitochondria. In common with the action of CC extract pretreatment of the fat body in vitro with 10(-5) M cyclic AMP, 10(-5) M 8-bromo-cyclic AMP, or 10(-5) M forskolin increased mitochondrial respiration approximately 30%. Octopamine (10(-4) M) elicited a response similar to that obtained with CC extract. Neither 10(-5) M cyclic AMP nor 10(-5) M 8-bromo-cyclic AMP stimulated respiration when applied directly to the mitochondria. These results suggest that the factor in CC extract manifests its effect intracellularly through the activation of a cyclic AMP-dependent protein kinase. This interpretation is also based on the finding that diamide, an inhibitor of protein kinase, inhibits CC-dependent and cyclic AMP-dependent mitochondrial respiration. The physiological role of the CC factor responsible is not known.

EVIDENCE THAT ECDYSIS IN THE LARVAL COCKROACH, Periplaneta americana L. IS TRIGGERED BY AN INCREASE IN THE CONCENTRATION OF HEMOLYMPH SUGAR

Ecdysis in insects can be defined as shedding of the cuticle at the end of a larval stadium. This event can only occur after the peak titer of ecdysteroid in the hemolymph has returned to a low level. In the cockroach Periplaneta americana, ecdysis is strongly correlated with a rise in the concentration of trehalose and glucose in the hemolymph, leading to the idea that a causal relationship may exist between both events. The objective in this study was to determine if an increase in hemolymph sugar level would shorten the time to ecdysis in cockroach larvae with experimentally delayed ecdysis. The last larval stadium of P. americana averages 33.5 days but this increases significantly if the larva is injected with a small volume of saline. Injection of 10 μl of saline on day 20 and on four successive days lengthened the stadium by as much as 2 weeks. If, however, trehalose or glucose is incorporated into the saline, approximately 40% of the treated larvae undergo ecdysis at the same time as uninjected larvae. Injection of Peram-AKH, the hypertrehalosemic hormone, also decreases the time for ecdysis to occur. This suggests that peak levels of ecdysteroid trigger the release of Peram-AKH, which then leads to activation of trehalose synthesis. The results support the hypothesis that elevated hemolymph sugar is a contributing factor in the removal of ecdysteroid from the hemolymph.