Comp. B&hem. Physiol. Vol. 118A, No. 4, pp. 1225-1231, 1997

Copyright 0 1997 Elsevier Science Inc. All rights reserved.

ELSEVIER

1SSN 0300-9629/97/$17.00

1’11 SO%D9629(97)00043-1

Hypertrehalosemic Hormones Increase the Concentration of Free Fatty Acids in Trophocytes of the Cockroach (Pev-@neta clmericana) Fat Body

Irshad Ali and John E. Steele DEPARTMENT OF ZOOLOGY,THE UNIVEFSITY OF WESTERN ONTARIO, LONDON, ONTARIO, CANADA N6A 5R7

ABSTRACT. 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. COMP BIOCHEM PHYSIOL 118A;4:1225-1231, 1997.

0 1997 Elsevier Science Inc.

KEY WORDS. Cockroach, corpus cardiacum, fat body, free fatty acid, hypertrehalosemic hormone, mycetocyte,

Periphetu americana, urate cell

INTRODUCTION

Cockroach fat body, as well as that of many other insects, contains large reserves of glycogen that can be accessed for the synthesis of trehalose which is subsequently released to the hemolymph. The rate of synthesis of trehalose from gly- cogen is increased by the action of the hypertrehalosemic hormones (HTH). These hormones also activate phospho- rylase, which suggests that this event is primarily responsible for the shift in metabolism that favors the synthesis of treha- lose from glycogen. However, other agents, including cyclic AMP, methyl xanthines, and the calcium ionophore A23187, also produce strong activation of phosphorylase (5), yet none of these has a significant effect on trehalose efflux from the fat body (5,lO). Clearly, the hypertrehalo- semic response requires more than phosphorylase activation alone.

In a previous study (Ali and Steele, unpublished data), various inhibitors of cyclooxygenase and phospholipase A! were shown to block HTH-activated trehalose synthesis in the intact fat body. Since cyclooxygenase is a key enzyme in the formation of prostaglandins from arachidonic acid, a common product of the phospholipase Az reaction, it is

Address repmu requests to: J. E. Steele, Department of Zoology, The Univer-

sity of Western Ontario, London, Ontario, Canada, N6A 5B7. Tel. (519)

661-3136; Fax (519) 661-2014. Received 14 October 1996; revised 27 March 1997; accepted 8 April

1997.

reasonable to propose that prostaglandins or related metab- olites may also be involved in the synthesis of trehalose or its release from the fat body. To determine whether metabo- lites of arachidonic acid might play a role in the hypertreha- losemic response, there was a need to know whether the concentration of arachidonic acid, or of fatty acids that could serve as substrates for arachidonic acid synthesis, might increase in response to HTH.

Intact fat body is an unsuitable system for unravelling the nuances of HTH action becauses it comprises three kinds of cells (1). These include trophocytes, urate cells, and my- cetocytes, of which only the trophocytes are considered to be involved in trehalose synthesis. Thus, the presence of the other cells precludes the accurate determination of fatty acids in the trophocytes. The present study employs a ho- mogeneous preparation of trophocytes to show that the ac- tion of HTH causes a significant change in the concentra- tion of certain free fatty acids within these cells.

MATERIAL AND METHODS Insects

The cockroaches, Periphneta americana, were reared in the laboratory at 28-30°C and 60 2 5% relative humidity with a photophase:scotophase for 12 hr each. Food (pulverized Purina Dog Chow’ combined with 10% sucrose, w/w, and water were supplied ad libitum. Only adult males, l-2 months old, were used. The cockroaches were neck-ligated in late afternoon for use the following morning. During this

1226 I. Ali and J. E. Steele

period, they were held in glass jars with the relative humid-

ity maintained at 90-95% to prevent dessication.

Reagents

Inorganic chemicals were of the highest grade available and

were obtained from various laboratory suppliers. Solvents

were purchased from British Drug Houses, Toronto, On-

tario, and Fisher Scientific Co., Toronto, Ontario, ~,cx,,cx-

Trifluoro-m-toluidine, methyl iodide, silver oxide, and

methyl propionate were purchased from Aldrich Chemical

Co., Milwaukee, WI. Fatty acids and fatty acid methyl esters

were supplied by Sigma Chemical Co., St Louis, MO, and

BIOMOL Research Laboratories, Plymouth Meeting, PA.

Collagenase (collagenase A) was obtained from Boehringer

Mannheim Canada, Laval, Quebec.

Physiological Saline

The physiological saline used for the disaggregation of the

fat bodies and as an incubation medium for the dispersed

trophocytes contained 215 mM NaCl; 4.8 mM KCl; 1mM

CaCl,; 1 mM MgCl,; 5 mM glucose; 40 mM trehalose; and

40 mM hepes buffer adjusted to pH 7.4 with 1 M NaOH.

The saline was aerated with filtered air just prior to use.

Hormones

Protein-free extracts of the corpora cardiaca were prepared

as previously described (8). A concentrated solution of

gland extract was prepared in physiological saline and stored

in polyethylene vials at -20°C. The synthetic hormones

HTH-I and HTH-II were obtained from Peninsula Labora-

tories, Belmont, CA, and were prepared as stock solutions

in 50% ethanol and stored in polyethylene vials.

Disaggregation of Fat Body

Fat bodies were disaggregated using collagenase, as de-

scribed previously (9), with the exception that the physio-

logical saline employed was that described in this study. For

each preparation of cells, the yield was determined using a

hemocytometer.

Incubation of Trophocytes RESULTS

Trophocytes, generally 50,000-60,000 in lml of saline were A series of preliminary experiments were performed to de-

pipetted into 40 ml polypropylene centrifuge tubes and in- termine which of the intracellular free fatty acids might be

cubated at 30°C for 30 min in a shaker water bath. Hor- affected by the known corpus cardiacum hormones. The

mone was then added and incubation of the cells allowed only free fatty acids that could be shown to respond to the

to continue for an additional 15 min. The reaction was hormones were palmitic, stearic, oleic, and linoleic acid.

stopped by pipetting the total incubation mixture into a The study, therefore, focussed on these four free fatty acids,

IO-ml plastic syringe barrel fitted with a 20-gauge needle which constituted the major part of the total free fatty acid

inserted through a rubber stopper and supported by a 250- pool. Usually, three additional fatty acids could be demon-

ml filter flask. The syringe contained a snug fitting disc of strated in the extracts but in low concentration which pre-

glass microfiber filter (Whatman GF/A) to retain the tro-

phocytes. Mild vacuum was applied to the flask to draw the

extracellular medium into the flask and away from the cells.

The filter containing the cells was quickly withdrawn from

the syringe with forceps and transferred to 1.5 ml of ice cold

methanol in a centrifuge tube. The tube was then flushed

with Nl and sealed with a teflon-lined screw cap. The sam-

ple was mixed thoroughly by vortexing and allowed to stand

for 20 min at 4°C. After centrifugation at 450 g for 10 min,

the supernatant was removed for analysis of the fatty acid

content as described below.

Measurement of Fatty Acids

Free fatty acids were separated and quantified by gas chro-

matography using “on column” methylation with tri-

methyl-( a,a,a-trifluoro-m-tolyl) ammonium hydroxide

(TMTFTH) (4). Briefly, the sample in 1.5 ml of methanol

was transferred to a glass stoppered centrifuge tube followed

by 5 ml of hexane and the mixture shaken vigorously. Fol-

lowing acidification by the addition of 0.5 ml of 1 M phos,

phoric acid to drive the fatty acids into the hexane phase

the sample was centrifuged at 450 g for 2 min. The lower

methanol phase was removed with a syringe and the hexane

phase washed twice with 2-ml aliquots of 0.1 M phosphoric

acid. The complete removal of the lower aqueous phase is

essential for efficient recovery of the fatty acids.

The methylated fatty acids were analyzed on a Hewlett-

Packard 571 IA gas chromatograph with a flame ionization

detector and a J&W DB-Wax capillary column (30 m X 0.53 mm i.d.). The splitless injection port and flame ioniza-

tion detector were maintained at 250°C and the oven pro-

grammed to operate from 190-230°C at a rate of 8”C/min.

Helium at 5 ml/min was used as the carrier gas. Data were

recorded using a Hewlett-Packard 3380A integrator. The

recovery of palmitic, stearic, oleic, and linoleic acids was

determined to be 95, 86, 85, and 90%, respectively.

Statistical Analysis

The significance of differences was tested using a t-test for

unpaired data, analysis of variance, and a multiple compari-

son test (Duncan) ( 11).

Free Fatty Ada in Prripheta 1227

u ._ l2 0 Control

2 lo- n HTH-I r z mz a-

LZ 0

Palmitic Stearic

A Oleic Linoleic

Fatty Acid

FIG. 1. The stimulatory effect of HTH-I on the free fatty acid content of dispersed trophocytes in vitro. Trophocytes (-50,000) in 1 ml of saline were preincubated at 30°C for 30 min. HTH-I, 100 pmol per ml, was added to one half of the samples and all of the samples incubated for an addi- tional 15 min. The total free fatty acids in the cells was deter- mined as described in the Materials and Methods. Analysis of the data reveals that the cellular content of palmitic, stea. tic, oleic, and linoleic acid was increased by the HTH-I (P < 0.001, n = 14).

eluded accurate measurement. The possibility that these

fatty acids might also have responded to the hormones can-

not be ruled out.

Incubation of trophocytes with HTH-I caused the intra-

cellular concentration of each of the free fatty acids investi-

gated to increase (Fig. I). A comparable effect was obtained

with HTH-II (Fig. 2). That these effects of the hormones

are a consequence of a specific interaction between the hor-

mones and receptors, presumably at the cell surface, is sug-

gested by the following experiment. A mixed population of

urate cells and mycetocytes derived from disaggregated tis-

sue, which initially contained the trophocytes, did not re-

spond to the same concentration HTH-I that produced a

significant response in the trophocytes (Fig. 3).

Our data show that corpus cardiacum extract containing

native HTH-I and HTH-II mimicks the stimulatory effect

of the synthetic hormones on free fatty acid concentration

but with one possible exception (Fig. 4). Palmitic acid levels

appeared to be unaffected by the gland extract. Because

each of the four fatty acids was determined on the same

sample resolved by the gas chromatograph, it is unlikely that

the failure to show a change in palmitic acid could be ex-

plained hy any mishap in handling of the sample. Further-

more, the concentration of corpus cardiacum extract used,

based on estimates of the amount of active material present

in the glands (7), would at the highest concentration em-

ployed be equal to or greater than that used ( 100 pmol) in

the experiments with the synthetic peptides Figs 1 and 2.

15.0 u 0 Control .-

: 12.5

1 n HTH-II

5

jz10.0

m2 i

Palmltic Stearlc Oleic Linoleic

Fatty Acid

FIG. 2. The increase in free fatty acid concentration of dis- persed trophocytes in vitro following a challenge by HTH- II. Trophocytes (-60,000) in 1 ml of saline were incubated as in Fig. 1 and the free fatty acids determined as described in Materials and Methods. The data show that HTH-II in. creased the content of each of palmitic (P < 0.025), steak (PC 0.005), oleic (P < 0.005), and linoleic acid (PC 0.005). For aU samples n = 4.

Control

HTH-I

sl Palmitic Stearic Oleic Linoleic

Fatty Acid

FIG. 3. Failure of synthetic HTH-I to increase the free fatty acid concentration in urate cells and mycetocytes in vitro. The cells, which constitute the sedimentable fraction ob- tained during the preparation of the trophocytes, were incu- bated in 1 ml of saline (- 120,000) in the same manner as were the trophocytes. The concentration of HTH-I was 100 pmol per ml. Analysis of the data did not reveal any differ- ences between the control samples and those treated with HTH-I (n = 5).

I. Ali and J. E. Steele

0.2 0.4 0.6

CC (GPE/mll

0.8 1.0

” / 0.0 012 ' 0.4 0.6 0.8 1.0

_I , 0 , I I /

0.0 0.2 0.4 0.6 0.8 1 .o

CC (GPE/ml) CC (GPE/ml)

0.2 0.4 0.6

CC (GPE/ml)

0.8 1.0

FIG. 4. Concentration dependent stimulatory effect of crude native HTH (CC) on the content of free fatty acids in dispersed trophocytes in v&o. Each sample contained -60,000 trophocytes in 1 ml of saline. The cells were preincubated for 30 min at 30°C followed by a further period of incubation during which half of the samples were incubated with corpus cardiacum extract containing the crude native HTH. The concentration of HTH employed is expressed as gland pair equivalents (GPE). Analysis of the data shows that the cellular content of steak, oleic, and linoleic acid was increased (P < 0.01; P < 0.05; P < 0.05, respectively). No effect on palmitic acid was demonstrated. For all samples D = 3.

The maximum concentration of oleic and linoleic acid was obtained with 0.2 gland equivalents. This amount of gland extract could be expected to contain approximately 20 and 8 pmol of HTH-I and HTH-II, respectively.

A maximal effect of HTH-II for three of the four fatty acids in the trophocytes was obtained with approximately 20 pmol of the hormone in 1 ml of cell suspension (Fig. 5). The increase was 27, 70, and 55% for palmitic, oleic, and linoleic acid, respectively. Stearic acid concentration in- creased 95% with 50 pmol HTH-II. The larger increase in palmitic and stearic acid obtained with 300 pmol of the hormone probably represents a pharmacological effect. The stimulatory action of the hormone is time dependent, at- taining its maximal effect after approximately 30 min (data not shown).

The fatty acyl activating effect of both HTHs suggests

that these hormones could facilitate the release of fatty acids to the hemolymph. To test this hypothesis, the effect of two concentrations of HTHs on the free fatty acid con- tent of the hemolymph was determined. The lower concen- tration of hormone was chosen because it was known to produce a strong hypertrehalosemic response (7). Although both hormones caused the hemolymph concentration of palmitic, stearic, oleic, and linoleic acid to increase mark- edly, the data show that HTH-II is considerably more active in this regard than is HTH-I (Fig. 6).

DISCUSSION

A comparison of the fatty acid concentration in the tropho- cytes with that of a mixed population of mycetocytes and urate cells shows, perhaps not surprisingly in view of the

Free Fatty Acids in Peripheta 1229

OJ, , I I I r I

0 50 100 150 200 250 300

HTH-II (Pmol/ml)

25

HTH-II (pmollml)

o-1, I I I , I 1 0

0 50 100 150 200 250 300

HTH-II (pmol/mli

I/

0 50 100 150 260 A0 360

HTH-II (pmol/mlI

FIG. 5. Concentration dependent stimulatory effect of synthetic HTH-II on the free fatty acid content of dispersed trophocytes in vitro Samples of trophocytes consisting of approximately 50,000 cells per ml of saline were incubated in the manner described in Materials and Methods. Free fatty acids were determined following 15 min incubation with the hormone. Analysis of the data reveals that the content of pahuitic, stearic, oleic and linoleic acid is elevated (P < 0.01, P < 0.001, P < 0.0005, P < 0.005 respectively). For all samples n = 5. Fifty pmol per ml of HTH-II appears to elicit a maximal increase of each fatty acid.

perceived function of the trophocytes, that the concentra-

tion of each measured fatty acid in the trophocytes is ap-

proximately double that in the mycetocytes and urate cells.

The data show that the concentration of free fatty acids in

the trehalose-producing trophocytes increases when these

cells are challenged with either HTH-I or HTH-II. That

this effect could be caused by the activation of a triacylglyc-

tential to serve as second messengers, either without modi-

fication or following their conversion to arachidonic acid,

a precursor of prostaglandins and other eicosanoids. Thus,

the study provides a basis for suggesting that fatty acids, or

their metabolites such as eicosanoids, may be an integral

part of the mechanism that regulates the production of tre-

halose by the fat body.

erol lipase seems most unlikely since corpus cardiacum ex- A mounting body of evidence suggests that fatty acids

tract, which contains both HTH-I and HTH-II, stimulates released from plasma membrane phospholipids in response

synthesis and not hydrolysis of triacylglycerol in the fat body to an extracellular signal may serve as second messengers for

(2). The increase in free fatty acids occurs only in the tro- various physiological mechanisms (3). For example, protein

phocytes, clearly demonstrating that the direct effect of kinase C is activated by arachidonic acid [for a review, see

HTH on the fat body is directed towards these cells. The (6)]. The possibility that fatty acids may be released from

four fatty acids increased by HTH, palmitic, stearic, oleic, phospholipids in the trophocyte plastna membrane to serve

and linoleic acid, are of interest because they have the po- a second messenger function is suggested hy the observation

1230 I. Ali and J. E. Steele

80 100 0 HTH-I

0 HTH-II

f

80

s -3 .E 60

x

.o k 40

Q G

20

0 0 0 20 40 60 80 100

Hormone (pmol/ml)

300 1

140 o HTH-I 1 0 HTH-I

0 HTH-II 120 1

l HTH-II

z 200

R 2 150

0

‘LE 100 0

50

- I

0 20 40 60 80 100

Hormone (pmol/mlI

HTH-I

HTH-II

I

0 I I I I I

20 40 60 80 100

Hormone (pmol/ml)

I , , I ,

20 40 60 80 100

Hormone (pmol/ml)

FIG. 6. Elevation of free fatty acid levels in cockroach hemolymph LD tivo following injection of HTH4 and HTH-II. The cockroaches were injected with hormone in a final volume of 10~1 of ethanol 95%: saline, 50:50, V/V: The concentration shown is that in the hemolymph and is an estimate based on an assumed hemolymph volume of 125 ~1. The controls received the same amount of carrier without hormone. After injection the cockroaches were returned to the insectary for 1 hr. The cockroaches were then anaesthetized by chiig for 10 min at - 16°C and the hemolymph removed by centrifugation at 27 g for 10 min. Free fatty acids were determined using 100 ~1 of hemolymph, which was treated in a manner identical to that used for the trophocytes. Analysis of the data showed that the concentration of pahnitic, steak, and oleic acid was increased significantly by HTH-I (P < 0.005; P < 0.005; P < 0.001, respectively, n = 5) as well as HTH-II (P < 0.001; P < 0.005; P < 0.0025, respectively, R = 5). Neither hormone had a significant effect on linoleic acid.

that inhibitors of phospholipase Ar block the HTH-stimu- lated release of trehalose from the trophocytes (Ali and Steele, unpublished studies). This interpretation, although possible, can only be regarded as tentative since the fatty acids released from the phospholipid also have the potential to be converted to prostaglandins.

The data suggest that a change in the concentration of free fatty acids could be linked to a regulatory function since they are evoked by physiological levels of HTH. A maximal effect of HTH-II on each of the fatty acids was obtained using 20 pmol . ml-‘. This value when prorated to account for an estimated hemolymph volume of 125 ,~l represents

approximately 2.4 pmol, well below the amount of 40 pmol of HTH-II estimated to be present in the corpus cardiacurn of Periplanem (7). The maximal effect of the hormone on fatty acid Levels requires a period of about 30 min. From this time onwards the rate of trehalose efflux from fat body due to the stimulatory effect of HTH is generally in decline (5). These events suggest that the increase in fatty acids and the decrease in the rate of trehalose efflux from the trophocytes may be linked.

The stimulatory effect of HTH-I and HTH-II on the fatty acyl content of the hemolymph is of interest for two reasons. First, at the higher concentration (100 pmol ml-‘), the

Free Fatty Acids in PeriplanetL1 1231

stimulatory effect of HTH-II was significantly greater than

that of HTH-I with respect to oleic acid and marginally

greater for each of the other fatty acids. However, at the

lower hormone concentration of 15 pmol ml-‘, HTH-I was

almost without effect, whereas the effect of HTH-II was

near maximal. These differences in the potencies of each

hormone are t)f some interest since they do not appear to

be reflected in their action on hemolymph trehalose (7).

Secondly, the results are also of interest because they suggest

that the trophocytes may generate fatty acids for export to

the hemolymph where they could subsequently be trans-

ported to peripheral tissues such as the musculature to serve

as a source of energy.

This research was supported in part by a Research Grant from the Natu- ral Sciences and Engineeering Research Council of Canada.

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