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

Peptides 24 (2003) 1545–1551

Lipid metabolism in the cockroach, Periplaneta americana, is activated by the hypertrehalosemic peptide, HTH-I Elisha Oguri, John E. Steele∗ Department of Biology, The University of Western Ontario, London, Ont., Canada N6A 5B7 Received 3 April 2003; accepted 31 July 2003

Abstract 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 6 h 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 2 h after injection of HTH-I and returns to normal at 6 h. 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. © 2003 Published by Elsevier Inc. Keywords: Fat body; Fatty acids; Hemolymph; Hypertrehalosemic hormone; Lipid metabolism; Neuropeptide; Neutral lipid; Phospholipase A2 ; Phospholipid

1. Introduction Corpus cardiacum extract containing the hypertrehalosemic hormones Pea HTH-I and Pea HTH-II (HTH-I, HTH-II) has been shown to cause a marked increase in hemolymph trehalose when injected into the hemocoel of the cockroach (Periplaneta americana) [24]. A similar response to the synthetic hormones has been demonstrated in other species of insects. Although the physiological function of HTH-I and HTH-II is poorly understood it has been linked to a need to supply a source of energy for muscle activity, and to generate substrates for biosynthetic reactions such as chitin synthesis, an integral step in the formation of the exoskeleton. The likelihood that the HTHs might serve a different function was alluded to in an early study showing that extracts of the corpus cardiacum of P. americana caused fat body triacylglycerol and diacylglycerol to increase while lowering the same neutral lipids in the hemolymph [6]. At the time, it was thought unlikely that this effect was due to the hypertrehalosemic factor in the crude gland extract although the possibility could not be ruled out. Very recently, we revisited this problem because synthetic Pea HTH-I and ∗

Corresponding author. Tel.: +1-519-661-3136; fax: +1-519-661-3935. E-mail address: [email protected] (J.E. Steele).

0196-9781/$ – see front matter © 2003 Published by Elsevier Inc. doi:10.1016/j.peptides.2003.07.016

Pea HTH-II had become available. Unequivocal evidence has now been obtained [19] which shows that both synthetic peptides have effects similar to those described for crude corpus cardiacum extract. Additionally, the hormones also increase free fatty acid levels in both the hemolymph and fat body and decrease the concentration of phospholipid in both tissue compartments [19]. The decrease in phospholipid in the hemolymph and fat body is of special interest, particularly with respect to the hemolymph, because in quantitative terms the concentration of this lipid in the hemolymph is not only high but is greater than that of diacylglycerol [4]. In this paper, our aim has been to offer additional evidence that HTH decreases phospholipid levels in the hemolymph and fat body using a different experimental approach than that used previously [19]. In addition, we have also examined the effect of HTH-I on the lipid composition of the fat body in vitro to determine whether any effects of the hormone in this situation are comparable to those found in vivo. 2. Materials and methods 2.1. Insects Adult male cockroaches, P. americana, 4–6 weeks after the final moult were used in the study and reared in an

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insectary at 28–30 ◦ C with a relative humidity of 60 ± 10% and a photocycle of 12 h light and 12 h dark. The diet contained 82% rolled oats, 5% yeast extract, 10% sucrose, and 3% peanut oil by weight. Both food and water were freely available. 2.2. Hormones and reagents The HTH-I used in this study was obtained from Peninsula Laboratories, Belmont CA. Conjugated linoleic acid (octadecadienoic acid: CLA), mepacrine and all standards were supplied by Sigma Chemical Co., St. Louis, MO. The EM Science silica gel, all solvents, and reagents were obtained from BDH, Toronto, Ontario. 2.3. Collection of tissue Hemolymph was collected by centrifugation. A ligature was tied around the posterior cervical region of the cockroach using a cotton thread and the mouth parts then sealed with molten wax to prevent contamination of the hemolymph with fluid from the gut. The prothoracic legs were severed at the coxa with sharp scissors and the cockroach inserted into a chilled 3 cm3 plastic disposable syringe. The syringe containing the cockroach was placed in a chilled 1.3 cm ×10 cm test tube and centrifuged in a clinical centrifuge at 750 rpm for 5 min. Hemolymph from three cockroaches was pooled to provide a single sample. If not used immediately, the hemolymph was quick frozen and stored at −20 ◦ C. The fat body was removed after addition of a couple of drops of physiological saline (154 mM NaCl; 8 mM KCl; 2 mM CaCl2 ; 3 mM MgCl2 ; and 40 mM Hepes buffer, pH 7.4) to the abdominal cavity to keep the tissues moist. The digestive tract was removed and the two lobes of fat body cut away from the inner wall of the body cavity and rinsed in saline prior to incubation. Incubations were performed in 2 ml of saline in a 20 ml scintillation vial at 30 ◦ C in a shaking water bath. In all experiments the fat body lobe used for the experimental treatment was compared with the untreated contralateral lobe taken from the same individual. Prior to the analysis of fat body the tissue was blotted dry on a ground glass surface to remove excess moisture and then weighed. Whenever tissue could not be extracted immediately it was frozen on dry ice prior to weighing and stored at −20 ◦ C. 2.4. Extraction of lipid Total lipid in hemolymph and fat body was extracted as follows. One hundred ␮l of hemolymph was transferred to a small Potter–Elvehjem homogenizer containing 0.5 ml of chloroform–methanol (1:1, v/v) and homogenized with 10 strokes of the pestle. The homogenate was transferred to a centrifuge tube and the homogenizer rinsed with 2× 0.5 ml of the same solvent mixture. The washings were combined with the original homogenate. After centrifuging the sample at 1500 rpm for 10 min in a clinical centrifuge, the su-

pernatant was transferred to a test tube and dried under a gentle stream of dry nitrogen. For fat body, one lobe of tissue was homogenized in 4 ml of chloroform–methanol (1:1, v/v) using a Potter–Elvehjem homogenizer. The sample was centrifuged at 1500 rpm for 5 min and 0.4 ml of the supernatant transferred to a test tube, dried under a gentle stream of nitrogen, and used for the analysis of lipid. 2.5. Separation of lipid classes Separation of lipid groups in hemolymph and fat body extracts was carried out on small silica gel columns [11] using silica gel that had not been washed with methanol as described in the original report. This method is designed to separate cholesterol esters, triacylglycerides, diacylglycerides, monoacylglycerides, non-esterified fatty acids and polar lipids such as phospholipids. Each lipid class was separated cleanly. All eluted lipid fractions, with the exception of fraction 1 containing cholesterol esters which were not included in the study, were dried using a rotary evaporator. 2.6. Free fatty acids Total fatty acids were determined using a colorimetric method involving formation of a complex of the fatty acid with copper and diphenylcarbazide [12]. The sample residue of fraction 4 from the silica gel column containing the free fatty acids was extracted twice with 2.5 ml of chloroform and both extracts combined in a 1.8 cm × 15 cm test tube for the colorimetric reaction. Palmitic acid was used as a standard and the free fatty acids expressed as palmitate equivalents. 2.7. Determination of acylglycerols and phospholipids Acylglycerol and phospholipid were determined indirectly by measuring the glycerol content of the parent molecule. The release of glycerol from acylglycerol and phospholipid and its subsequent determination are described in detail elsewhere [9]. This method involves liberation of the glycerol backbone by transesterification and its subsequent conversion to a colored derivative for estimation by spectrophotometry. The samples of tri-, di-, and monoacylglycerol eluted from the silica gel column, from which the solvents had been evaporated, were extracted with 0.5 ml of heptane. The dried phospholipid samples were extracted with 0.5 ml of isopropanol. From each sample, 0.4 ml was transferred to a test tube and the sample made up to 0.5 ml with heptane for transesterification and determination of glycerol [9]. Neutral lipid and phospholipid standards were carried through the entire procedure for each batch of samples. 2.8. Determination of glycerol 3-phosphate A lobe of fat body was placed in a microcentrifuge tube containing 1 ml of 0.5 M HClO4 and homogenized by

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sonification for 10 s. After centrifugation at 10,000 × g for 10 min, the pH of the supernatant was adjusted to 7.0 with 5 M K2 CO3 . The sample was re-centrifuged and 1 ml removed for analysis. The assay mixture contained 1 ml of buffer (400 mM hydrazine, 1 M glycine, and 5 mM EDTA adjusted to pH 9.5); 100 ␮l of 60 mM NAD and 1 ml of sample [16]. 2.9. Phospholipase A2 assay The phospholipase A2 activity of hemolymph was estimated fluorometrically with the pyrene-labeled phospholipid, 1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3phosphocholine as the substrate, in the presence of serum albumin [21,26]. The reaction was started by the addition of 20 ␮l of hemolymph to the cuvette and the fluorescence recorded every 5 s for 400 s at 30 ◦ C. 2.10. Data analysis The data were analyzed for significance of differences with ANOVA in conjunction with Duncan’s multiple comparison test, and a t test for unpaired data using a software package (Stat-100) supplied by Biosoft®.

3. Results Although HTH-I has been shown to lower total phospholipid levels in cockroach hemolymph and fat body [19], it was not known whether this effect of HTH-I was limited to particular phospholipids in either tissue. The major phospholipids present are phosphatidylcholine and phosphatidylethanolamine which together constitute nearly 80% of the total (unpublished data). In response to HTH-I fat body phosphatidylcholine decreased 34% whereas phosphatidylethanolamine decreased 46% (Table 1). In the hemolymph, both phospholipids

Table 1 Effect of HTH-I on phospholipid in the hemolymph and fat body of P. americana Metabolite

Tissue

Treatment Control

PE PE PC PC

(␮mol/ml) (␮mol/g) (␮mol/ml) (␮mol/g)

Hemolymph Fat body Hemolymph Fat body

1.01 4.89 1.41 6.06

± ± ± ±

HTH-I 0.07a 0.26a 0.11a 0.73a

0.57 2.65 0.83 4.02

± ± ± ±

0.08b 0.31b 0.07b 0.30b

The cockroaches were injected with 100 pmol of HTH-I. The tissues were removed 6 h later and the phospholipid estimated as glycerol released from the parent molecule. Abbreviations used: PE, phosphatidylethanolamine; PC, phosphatidylcholine. The data were analyzed using a t-test for unpaired data. For each pair of values, a significant difference (P < 0.05) is indicated by different letters.

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Table 2 Stimulatory effect of HTH-I on the appearance of phospholipid metabolites Metabolite

Tissue

Treatment Control

HTH-I

GPC (␮mol/ml or g)

Hemolymph Fat body

0.97 ± 0.41 ± 0.04a

1.49 ± 0.09b 0.69 ± 0.04b

Choline (␮mol/ml or g)

Hemolymph Fat body

0.32 ± 0.03a 0.66 ± 0.12a

0.24 ± 0.02b 0.49 ± 0.10a

Glycerol (␮mol/ml or g)

Hemolymph Fat body

0.03 ± 0.002a 0.59 ± 0.04a

0.04 ± 0.004a 1.33 ± 0.21b

0.08a

The cockroaches were injected with 100 pmol of HTH-I. The tissues were removed 6 h later and the metabolites estimated enzymatically as described in Section 2. Abbreviation used: GPC, glycerophorylcholine. Values shown are means ± S.E.M. (n = 6). The data were analyzed using a t-test for unpaired data. For each pair of values, a significant difference (P < 0.05) is indicated by different letters.

were decreased by HTH-I within the range of 41–44% (Table 1). The decrease in phospholipid induced by HTH-I could have occurred because of a lowered rate of synthesis or, alternatively, an increase in the rate of catabolism leading to an increase in production of phospholipid metabolites. The data show that the second alternative is more probable (Table 2). HTH-I increased the concentration of glycerophosphorylcholine in the hemolymph and fat body by 54 and 68%, respectively whereas glycerol in the fat body increased by 125%. Interestingly, the concentration of choline is decreased in both tissue compartments by 25%. The increased formation of glycerophosphorylcholine in hemolymph owing to the action of HTH-I could be explained on the basis of an increase in the activity of phospholipase. To test this possibility, the activity of phospholipase in the hemolymph at different times after injection of HTH-I was determined. The results (Fig. 1) show clear evidence of an increase in phospholipase activity which peaked approximately 2 h following injection of the hormone and then returned to the resting level at 6 h, the time at which most of the lipid analyses have been made. Understanding the mechanism of action of HTH-I on lipid metabolism would be greatly facilitated if the hormone could be shown to elicit in vitro not only the customary stimulatory effect on triacylglycerol accumulation in the fat body, but also the change in the other lipid classes as well. To do this, paired fat body lobes were incubated with and without HTH-I. Analysis of the tissue lipids shows that the response to HTH-I is similar to that of the fat body in the intact cockroach (Table 3). Worthy of note, however, is the fact that HTH-I did not have a stimulatory effect on the accumulation of triacylglycerol in the fat body under these conditions. Although the diminished level of phospholipid in HTH-I-treated cockroaches suggests that this is the most likely source of fatty acids for the synthesis of triacylglyc-

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Fig. 1. Activation of hemolymph phospholipase by HTH-I. Each cockroach was injected with 10 ␮l of physiological saline containing 100 pmol of HTH-I or saline alone. The hemolymph was removed after injection at the times shown in the figure and the phospholipase activity determined immediately as described in the Section 2. Analysis of the data shows that differences due to HTH-I treatment between 30 min and 4 h inclusive are significant (P < 0.01). The values shown are means ± S.E.M. (n = 6).

erol this does not preclude the possibility that they might also arise de novo from carbohydrate. To test the possibility that fatty acids might have been generated from carbohydrate, fat bodies were incubated in vitro with and without CLA, both in the presence and absence of HTH-I. CLA is a potent inhibitor of fatty acid synthesis [20] and would be expected to block the appearance of free fatty acids, as well

as newly formed diacylglycerol, if the acyl groups were supplied by synthesis from acetylCoA. The results (Table 4) indicate that there is no interference with the appearance of free fatty acids, nor of those required for the higher rate of diacylglycerol synthesis induced by HTH-I. We conclude from these data that the additional free fatty acids and those esterified in the newly formed diacylglycerol, as a result of treating the fat body with HTH-I, do not arise from a non-lipid precursor. The preceeding data support the idea that within the fat body the fatty acids destined for incorporation into diacylglycerol, and ultimately into triacylglycerol, are channeled through phospholipid. To test this view, the effect of the phospholipase A2 inhibitor mepacrine on the lipid concentration in HTH-I-treated fat body was determined (Table 5). Mepacrine, acting alone, had no effect on the individual lipid levels but together with HTH-I, which normally depletes phospholipid in the fat body, completely blocked the action of the hormone so that the phospholipid remained at the resting level. Coincident with this effect, the rise in free fatty acid levels normally induced by HTH-I was blocked as was the increase in diacylglycerol. Glycerol 3-phosphate, which serves as the skeleton for the attachment of fatty acids to form acylglycerols and phospholipids, may be derived from glycogen or glucose via the glycolytic pathway. Alternatively, it may also originate from glycerol which undergoes phosphorylation by glycerol kinase which is present in the insect fat body [31]. To determine whether activation of lipid metabolism in the fat body by HTH-I is accompanied by an increase in glycerol 3-phosphate cockroaches were injected with 100 pmol of

Table 3 Effect of HTH-I on lipid in Periplaneta americana fat body in vitro Treatment

Metabolite (␮mol/g) TAG

Control HTH-I

DAG

402 ± 416 ± 16a

MAG

10.3 ± 13.4 ± 0.57b

14a

PL

0.74 ± 0.51 ± 0.06b

0.47a

0.04a

FFA

9.41 ± 6.40 ± 0.52b 0.75a

1.83 ± 0.15a 3.44 ± 0.28b

Paired fat body lobes were incubated individually in 2 ml of physiological saline at 30 ◦ C for 1 h. Where indicated the medium contained 50 pmol of HTH-I/ml. The lipid fractions were separated and estimated as described in Section 2. Abbreviations used: TAG, triacylglycerol; DAG, diacylglycerol; MAG, monoacylglycerol; PL, phospholipid; FFA, free fatty acids. Values shown are means ± S.E.M. (n = 6). The data were analyzed using a t-test for paired data. For each pair of values, a significant difference (P < 0.01) is indicated by different letters. Table 4 Failure of conjugated linoleic acid to block the effect of HTH-I on lipid metabolism in the fat body of Periplaneta americana in vitro Treatment

Metabolite (␮mol/g)

Control CLA HTH-I HTH-I + CLA

367.3 368.7 386.1 401.1

TAG

DAG ± ± ± ±

27.6a 23.0a 18.4a 13.8a

9.00 9.40 11.98 10.96

MAG ± ± ± ±

0.43a 0.34a 0.75a 0.59a

0.74 0.56 0.48 0.49

± ± ± ±

PL 0.04a 0.03b 0.07a 0.04a

9.50 9.10 6.63 5.86

FFA ± ± ± ±

0.74a 0.78a 0.69a 0.64a

2.15 2.77 3.85 3.26

± ± ± ±

0.16a 0.39a 0.25a 0.43a

Paired fat body lobes were incubated individually in 2 ml of physiological saline at 30 ◦ C for 1 h. Where indicated, the medium contained 50 pmol of HTH-I/ml and/or 10 ␮g CLA/ml. The lipid fractions were separated and estimated as described in Section 2. The data sets control vs. CLA and HTH-I vs. HTH-I + CLA were analyzed independently using a t-test for paired data. Abbreviation: CLA, conjugated linoleic acid; other abbreviations as in Table 3. The effect of HTH-I vs. control, which did not utilize paired tissue, was analyzed using a t-test for unpaired data. HTH-I has an effect comparable to that shown in Table 3. Values shown are means ± S.E.M. (n = 6). For each pair of values, a significant difference (P < 0.05) is indicated by different letters.

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Table 5 Activation of lipid metabolism in Periplaneta americana fat body by HTH-I in vitro is blocked by mepacrine Treatment

Metabolite (␮mol/g) TAG

Control MEP HTH-I HTH-I + MEP

367.3 358.1 417.0 401.3

DAG ± ± 30.2a ± 36.4a ± 38.2b 27.6a

MAG

9.0 ± 8.51 ± 0.51a 13.75 ± 0.44a 8.71 ± 0.49b 0.43a

0.74 0.77 0.43 0.62

PL

± ± 0.05a ± 0.08a ± 0.06b 0.04a

9.50 9.42 7.95 10.95

FFA ± ± 0.61a ± 0.46a ± 0.53b 0.74a

2.15 2.07 3.94 2.31

± ± ± ±

0.17a 0.14a 0.37a 0.18b

Paired fat body lobes were incubated individually in 2 ml of physiological saline at 30 ◦ C for 1 h. Where indicated, the medium contained 50 pmol of HTH-I/ml and/or 1 mM mepacrine. The lipid fractions were separated and estimated as described in Section 2. The data sets control vs. MEP and HTH-I vs. HTH-I + MEP were analyzed independently using a t-test for paired data. Abbreviation: MEP, mepacrine; other abbreviations as in Table 3. Comparison of the effect of HTH-I vs. control, which did not utilize paired tissue, was analyzed using a t-test for unpaired data. The effect of HTH-I vs. control is comparable to that shown in Table 3. Values shown are means ± S.E.M. (n = 6). For each pair of values, a significant difference (P < 0.05) is indicated by different letters.

HTH-I and the fat bodies removed for analysis 6 h later. The results show that HTH-I caused the concentration of glycerol 3-phosphate to increase from 1.26 ± 0.09 ␮mol/g to 2.20 ± 0.15 ␮mol/g (P < 0.005; n = 6). 4. Discussion This study shows that HTH-I alters the neutral lipid, phospholipid and free fatty acids of fat body in vitro in a manner similar to that demonstrated for fat body in the intact cockroach [19], but with one important difference; HTH-I failed to increase the concentration of triacylglycerol. There are two pathways for the synthesis of triacylglycerol in the cockroach fat body: the monoacylglycerol acyltransferase pathway with relatively low activity [10] and the glycerol 3-phosphate pathway [5,28], originally described for mammals by Kennedy [13], and quantitatively the most important. The importance of glycerol 3-phosphate in triacylglycerol synthesis via this pathway is underscored by the fact that this intermediate is present in high concentration in the fat body [23], both in vitro and in vivo, and is increased by HTH. The failure of HTH-I to stimulate triacylglycerol synthesis in the fat body in vitro is probably related to the fact that in this state the tissue is no longer bathed in hemolymph which is especially rich in diacylglycerol and phospholipid. In vivo, HTH depletes hemolymph of part of its diacylglycerol and phospholipid [19] which suggests that this is a likely source of fatty acids for triacylglycerol synthesis in the fat body. In the absence of hemolymph in in vitro experiments, there would then be insufficient flux of fatty acids through the system to support the synthesis of measurable quantities of triacylglycerol. Although a change in free fatty acids, phospholipid and diacylglycerol in vitro occurs in the absence of hemolymph, indicating that HTH-I has a direct effect on the fat body, the stimulatory effect of the hormone on triacylglycerol synthesis in the fat body can only manifest itself when hemolymph is present to serve as a source of fatty acids. It could be argued that the effect of HTH described in this study is pharmacological rather than physiological because

of the high concentration of hormone used. A high concentration was used to ensure that the concentration would be well above threshold for the entire duration of the experiment. This approach seems justified in the light of recent experiments (Garcha and Steele, unpublished data) which show that several injections of a much smaller amount of HTH will produce an effect similar to that of the higher concentration. The additional free fatty acids in fat body treated with HTH-I, as well as the fatty acids incorporated into diacylglycerol, could arise de novo from a non-lipid precursor. Carbohydrate is a potential source since glycogen is rapidly degraded in the presence of the hormone [17]. Under this scenario acetylCoA arising from glucosyl residues would be converted into fatty acids by the fatty acid synthase system. That this is unlikely is shown by the failure of CLA, a potent inhibitor of fatty acyl synthase [20,18], to block the accumulation of free fatty acids and diacylglycerol in the HTH-treated fat body. Although fatty acids incorporated into diacylglycerol and ultimately into triacylglycerol are unlikely to arise from carbohydrate the glycerol to which the fatty acids are attached very likely is derived from glycerol 3-phosphate originating from glycogen. Interestingly, the concentration of glycerol 3-phosphate in fat body treated with HTH-I is two-fold higher than the control values [23]. The finding that the additional free fatty acids, and those incorporated into diacylglycerol in the HTH-I-treated fat body, do not arise de novo is consistent with the idea that they exist in an esterified form prior to treatment with the hormone. The robust decline in the level of phosphatidylcholine and phosphatidyethanolamine, which together comprise approximately three-quarters of the total phospholipid pool in the fat body [7], indicates their potential importance as a source of fatty acids. The concentration of both phospholipids is decreased by HTH-I, thus favoring the idea that phospholipid is an important target of HTH-I. The catabolism of phospholipid in the fat body, initiated by HTH-I, is accompanied by an increase in the concentration of free fatty acids and diacylglycerol. This increase in free fatty acids coincides with a rise in the concentration of glycerophosphorylcholine and suggests that the fatty acids are removed from the parent molecule by phospholipase activity and then become avail-

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able for incorporation into diacylglycerol and subsequently triacylglycerol. This outcome could arise in different ways. Both fatty acids could be removed simultaneously by a phospholipase B [8,14]or, alternatively, there could be step-wise deacylation by phospholipase A2 to remove the fatty acid in the sn-2 position followed by the removal of the fatty acid in the sn-1 position by a phospholipase A1 [30]. In this sense, the situation may be comparable to that in osmotically regulated renal medullary cells [15]. Unfortunately, very little is known of the full range of phospholipases in the fat body. The elevated level of glycerophosphorylcholine in the fat body of the intact cockroach following injection with HTH-I is probably a consequence of the increase in phospholipid catabolism [19] and could reasonably be expected to also occur in vitro. The subsequent catabolism of glycerophosphorylcholine in the fat body cannot be described with certainty but it seems likely that this might be mediated by the action of GPC:choline diesterase as occurs in rat medullary collecting duct cells [1]. This would lead to the formation of glycerol 3-phosphate and choline. Our data show that there is a large increase in the concentration of glycerol 3-phosphate in the fat body of cockroaches injected with HTH-I as occurs in vitro with fat body treated with corpus cardiacum extract [23]. Strangely, choline in the fat body is decreased following injection of the insect with HTH-I; possibly an indication that HTH-I stimulates metabolism of the choline formed. At the same time glycerol increases in both the hemolymph and fat body although this occurs predominantly in the fat body. Whether the glycerol is derived from glycerol 3-phosphate by the action of an acid phosphatase such as that described for eggs of Bombyx mori [3] remains to be determined. It is assumed that the catabolism of phosphatidylethanolamine will probably not differ markedly from that of phosphatidylcholine. The higher level of glycerophosphorylcholine and free fatty acids in the fat body following treatment of the cockroach with HTH-I points strongly to an increase in phospholipase activity as being the initiator of phospholipid mobilization. This conclusion is in keeping with the finding that phospholipase A2 activity in cockroach fat body, is activatable by HTH-I and HTH-II, but is restricted to the trophocytes within that tissue [25–27]. Catabolism of phosphatidylcholine and phosphatidylethanolamine in the hemolymph also appears to follow a similar pattern. The hemolymph enzyme, however, appears to differ from that in the fat body with respect to stability; dilution of the hemolymph in the assay medium causing the activity to be rapidly lost (unpublished data). Mepacrine, an inhibitor of mammalian phospholipase A2 [2], blocked the regulatory effect that HTH-I has on the free fatty acids, phospholipid and diacylglycerol in the fat body; an effect attributed to inhibition of phospholipase. This is not proof that the enzyme inhibited by mepacrine is a true phospholipase A2 . Indeed, the fact that less than one-quarter of the phospholipase activity in trophocytes is inhibited by phospholipase A2 inhibitors such as mepacrine

and 4-bromophenacyl bromide [27] casts considerable doubt on the likelihood that a phospholipase A2 is solely involved in the deacylation of the phospholipid. There is no reason to believe that anything other than removal of both fatty acids from the phospholipid occurs. The method used to estimate removal of fatty acids from the parent phospholipid molecule is sensitive only to the loss of fatty acids from the sn-2 position and gives no information about the possible release of fatty acids from the sn-1 position. It is possible therefore that two phospholipases may be involved in the removal of the fatty acids, one specific for the sn-2 position and the other for sn-1. If this scenario is correct the phospholipase A2 may be comparable to that described for the hemocytes [22] and fat body [29] of Manduca sexta. Alternatively, it is possible that a phospholipase B type enzyme is responsible for removal of both fatty acids from the phospholipid in the fat body and the hemolymph. Conventionally, HTH-I has been regarded as having an important function in mobilizing the carbohydrate reserves in cockroach fat body to supply energy as well as substrate for biosynthetic reactions. This view of the role played by the hormone must now be revised to include regulation of lipid metabolism, possibly for the purpose of promoting the storage of lipid in the fat body.

Acknowledgments This work was supported by a Research Grant from the Natural Sciences and Engineering Research Council of Canada (NSERC) to J.E.S. The Government of India generously supported E.O. with the award of a scholarship for postgraduate studies. References [1] Bauernschmidt HG, Kinne RKH. Metabolism of the ‘organic osmolyte’ glycerophosphorylcholine in isolated rat inner medullary collecting duct cells. I. Pathway for synthesis and degradation. Biochim Biophys Acta 1993;1148:331–41. [2] Chang HW, Kudo I, Tomita M, Inoue K. Purification and characterization of extracellular phospholipase A2 from peritoneal cavity of caseinate-treated rat. J Biochem 1987;102:147–54. [3] Chino H. Enzymatic pathways in the formation of sorbitol and glycerol in the diapausing egg of the silkworm, Bombyx mori. II. On the phosphatases. J Insect Physiol 1961;6:231–40. [4] Chino H, Katase H, Downer RGH, Takahashi K. Diacylglycerolcarrying lipoprotein of hemolymph of the American cockroach: purification, characterization, and function. J Lipid Res 1981;22: 7–15. [5] Downer RGH. Lipid metabolism. In: Kerkut GA, Gilbert LI, editors. Comprehensive insect physiology, biochemistry and pharmacology, vol. 4. Oxford: Pergamon Press; 1985. p. 77–113. [6] Downer RGH, Steele JE. Hormonal stimulation of lipid transport in the American cockroach. Periplaneta americana. Gen Comp Endocrinol 1972;19:259–65. [7] Fast PG. Insect lipids. Prog Chem Fats Lipids 1971;11:179–242. [8] Gassama-Diagne A, Fauvel J, Chap H. Purification of a new, calcium-independent, high molecular weight A2 /lysophospholipase

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[9]

[10]

[11]

[12] [13] [14]

[15]

[16]

[17]

[18]

[19]

(phospholipase B) from guinea pig intestinal brush-border. J Biol Chem 1989;264:9470–5. Giegel JL, Ham AB, Clema W. Manual and semi-automated procedures for measaurement of triglycerides in serum. Clin Chem 1975;21:1575–81. Hoffman AGD, Downer RGH. End product specificity of triacylglycerol lipases from intestine, fat body, muscle and haemolymph of the American cockroach, Periplaneta americana L. Lipids 1979;14:893–9. Ingalls ST, Kriaris MS, Xu Y, DeWulff DW, Tserng K-Y, Hoppel CL. Method for isolation of non-esterified fatty acids and several other classes of plasma lipids by column chromatography on silica gel. J Chromatogr 1993;619:9–19. Itaya K. A more sensitive and stable colorimetric determination of free fatty acids in blood. J Lipid Res 1997;18:663–5. Kennedy EP. Biosynthesis of complex lipids. Fed Proc 1961;20: 934–40. Kuwabara Y, Maruyama M, Watanabe Y, Tanaka S, Takakuwa M, Tamai Y. Purification and some properties of membrane-bound phospholipase B from Torulaspora delbrueckii. J Biochem 1988;104:236–41. Kwon ED, Jung KY, Edsall LC, Kim H-Y, Garcia-Perez A, Burg MB. Osmotic regulation of synthesis of glycerophosphocholine from phosphatidylcholine in MDCK cells. Am J Physiol 1995;268: C402–12. Lang GL. (−)-Glycerol 3-phosphate In: Bergmeyer HU, editor. Methods of enzymatic analysis, vol. 6. Weinstein: Verlag Chemie; 1984. p. 525–31. Lee Y-H, Keeley LL. Intracellular transduction of trehalose synthesis by hypertrehalosemic hormone in the fat body of the tropical cockroach, Blaberus discoidalis. Insect Biochem Mol Biol 1994;24:473–80. Loor JJ, Herbein JH. Exogenous conjugated linoleic acid isomers reducine bovine milk fat concentration and yield by inhibiting de novo fatty acid synthesis. J Nutr 1998;128:2411–9. Oguri E, Steele JE. A novel function of cockroach (Periplaneta americana) hypertrehalosemic hormone: translocation of lipid from hemolymph to fat body. Gen Comp Endocrinol 2003;132:46–54.

1551

[20] Pariza MW, Ha YL, Benjamin H, Sword JT, Gruter A, Chin SF, et al. Formation and action of anticarcinogenic fatty acids. Adv Exp Med Biol 1991;289:269–72. [21] Radvanyi F, Jordan L, Marie FR, Bon C. A sensitive and continuous fluorometric assay for PLA2 using pyrene-labelled phospholipids in the presence of serum albumin. Anal Biochem 1989;177: 103–9. [22] Schleusener DR, Stanley-Samuelson DW. Phospholipase A2 in hemocytes of the tobacco hornworm, Manduca sexta. Arch Insect Biochem Physiol 1996;33:63–74. [23] Sevala VL, Steele JE. Regulation of glycolytic intermediates in cockroach fat body by the corpus cardiacum. J Comp Physiol B 1991;161:349–55. [24] Steele JE. The site of action of insect hyperglyacemic hormone. Gen Comp Endocrinol 1963;3:32–46. [25] Sun D, Steele JE. Characterization of cockroach (Periplaneta americana) fat body phospholipase A2 activity. Arch Insect Biochem Physiol 2002a;49:149–57. [26] Sun D, Steele JE. Regulation of phospholipase A2 activity in cockroach (Periplaneta americana) fat body by hypertrehalosemic hormone: evidence for the participation of protein kinase C. J Insect Physiol 2002;48:537–46. [27] Sun D, Steele JE. Control of phospholipase A2 activity in cockroach (Periplaneta americana) fat body trophocytes by hypertrehalosemic hormone: the role of calcium. Insect Biochem Mol Biol 2002;32:1133–42. [28] Tietz A. Studies on the biosynthesis of diglycerides and triglycerides in cell free preparations of the fat body of the locust, Locusta migratoria. Isr J Med Sci 1969;5:1007–17. [29] Uscian JM, Stanley-Samuelson DW. Phospholipase A2 activity in the fat body of the tobacco hornworm Manduca sexta. Arch Insect Biochem Physiol 1993;24:187–201. [30] Waite M. Phospholipases. In: Vance D, Vance J, editors. Biochemistry of lipids, lipoproteins and membranes. Amsterdam: Elsevier Science; 1991. p. 269–5. [31] Zebe E. Die verteilung von enzymen des fettstoffwechels im Heuschreckenkörper. Verh Dtsch Zool Ges Münster/Westf 1959:309–14.