Gluconeogenesis in hepatopancreas of Chasmagnathus granulata crabs maintained on high-protein or carbohydrate-rich diets

ISN 0300~9629/47/$17.00 PlI SC7~@0-9629(97)L70241-7

ELSEVIER

Gluconeogenesis in Hepatopancreas of Chasmagnathus gran~k~taCrabs Maintained on High-Protein or Carbohydrate-Rich Diets Guenddinu

T. Oliveira and Roselis S. M. Da Siha

DEPARTAMENTODE FISIOLOGIA,INSTITUTO DE BIO&NCIAS, UNIVERSIDAL>E FEDERALDO RIO GRANDE DO SUL, 90050-l 70, PORTO ALEGRE, RS, BIIASII

ABSTRACT.

The

capacity

phosphoenolpyruvate to obtain

more

for glucose

carboxykinase

information

on the incorporation in Chasmapthus

or “C-lactic

pnulata,

acid, and its phosphoenolpyruvate

hydrate

diets.

recovered

from

pathway. cytosol

amount

“C-lactic

by glucose,

granulata

we investigated

glucose

in Chasmag~thus

activity

The data

activity

suggest

found

that

Inc.

KEY WORDS.

Crabs, diets, gluconeogenesis,

protein,

are fed. In animals

and lipid contents

fed normally

balanced

gluconeogenic

capacity

genie

are low in the fed state,

However,

fed high-protein

in mammals, diets,

mals and decrease

and increase

enzymes

of decapods

capacity

(2 1). It has been

food deprivation

has long been

(35),

that

other

en-

metab-

this tissue might

although

occurs

glucose

be

authors

have proposed that the hemocytes (23,24), the gills (46), or even ab&>minal muscles (13) could act in this capacity.

or carbo-

higher

than

that

for the gluconeogenic

the mltochondrial (10,000 X g) and from “C- a 1anine in the hepatopancreas

diet is equally

FEPCK,

subject

“C-&nine,

have

to end-point

across species.

“C-lactic

been

In Callinectes

carboxykinase

ity in hepatopancreas.

In contrast, levels

any significant

tase (FBPase)

and PEPCK specie>

(PC),

Lallier and Walsh

fructose-

of PEPCK

location,

hepatopancreas,

found

(29). Thus and

ac-

hand,

and

crustaceans. activity

measurable

in the white

muscle

that this tissue may he rttspon-

and pathway

variability

enzymes 1,6-bisphospha-

(29) found low PEPCK

and FBI&e

(29) activ-

et ul. (19) did not

or terrestrial

supidus gills. On the other

sible for gluconeogenesis interspecies

Henry

in muscle,

of aquatic

of Callinectes sapidus indicate pability,

significant

(PEPCK)

of gluconeogenic

carboxylase

gills of three

activities

to have

sapidus, Lallier and Walsh

detect

Similarly,

;lcid. glucose

documented

found phosphoenolpyruvate

(pyruvate

inhihi-

1997. Q 1997

for glycolytic enzymes, but the presence enzymes activities shows some variability

the gluconeogenic

m crustaceans

is still

uncertain

show

cagreat

( 17,29,37,

38, 46). Chasmqnathus panulata is a semiterrestrial crab that lives in the mesolittoral and supmlittoral zones of estuaries along the south Brazilian coast (4). Its alimentary behavior can be classitied as opportunistic ( 1 1). P revious studies showed that Chasmquthus

Rrcr~ve~l 25 N<)vemher 1996; revised 29 Aprd 1997; accepted 26 June 1997.

in vertc-

into

gluconeogenesis

tivity

dirts

fnlrn alanine

by high-prr)tein

concentration

in Callinectes

thought

of digestive

for lipid and carbohydrate suggested

birds and ac-

are high in the fed ani-

not only as a site for secretion

site of gluconeogenesis

during

fishes, and carnivorous

the gluconeogenic

zymes, hut also as a center the

of key gluconeo-

or stay the same during

(9,25,49,34). The hepatopancreas

in the diet they

diets, the maximal

and the activities

tivity of key gluconeogenic

olism

was detected

c>f different

to what

was significantly

levels of activity of gluconeogenic

pattern of metabolic adjustment to energy metabolism in an animal may undergo changes according to variations

to function

was not influenced “C-alanine

with

In order

granularc~ hrpatopancreas,

to synthetize

is not a saturated

hepatopancreas,

The

fasting.

from

the effecn

In contrast

Crustaceans

in carbohydrate,

associated

COMP BKXHEM PHYSIOL 118A;4:1429-1435,

INTRODUCTION

enzymes

activity,

or carbohydrate-rich

in vertebratex.

Elsevier Science

in this organ.

of the hepatopancreas

recovered

fed a high-protein

as has heen

in crabs,

carhoxykinase

carboxykinase

X g) fractions.

hepatopancreas,

capacity

capacity

glucose

granulata

of this tissue as a site for gluconetpntzbis.

acid into

and 40 mM of alanine

PhosFhocnolpyruvate (100,000

from Chasmnpthus tion

of lahelled acid,

in favor

carboxykinase the intrinsic

or lactic

The

in Chasmagnathus

argues

on the gluconeogenic

of 14C-alanine


synthesis

activity,

metabolic adjustment to the carbohydrate

grunulatu

had a characteristic

pattern

of

of carbohydrate and lipids according and protein Ievcls in the diet (27).

G. T. Oliveira and R. S. M. Da Silva

1430

Moreover,

the

in response ferent

carbohydrate

to fasting

diets

that

lipid

varied

the animals

metabolic

according

were previously

pattern

to the

dif-

submitted

to

EXPERIMENT

HP-fed with

animals 0.2

,&i

L-alanine

(50). In order

to obtain

genie capacity

more

information

or carbohydrate-rich

tion of 14C-alanine

or “C-lactic

diets on the incorpora-

acid into glucose

slices of Chasmugnathus

the PEPCK

on the gluconeo-

in vitro, the effects

in crabs, we investigated,

of high-protein pancreas

and

also

activity

on high-protein

granulata.

in hepatopancreas

We also report

of crabs maintained

or carbohydrate-rich

under

of [U-‘4C]-alanine

slices from

the above conditions

and

5 mM

unlabeled

He-

EXPERIMENTZ:SUBSTRATEDOSE-RESPONSECURVES.

slices from HP- or HC-fed

(120 min)

L-alanine

with graded

or lactic-acid

of [U-14C]-alanine the unlabelled

diets.

Hepatopancreas

were incubated

for 30, 60, 120, and 150 min.

patopancreas bated

in hepato-

I:TIMECURVE.

animals

concentrations

were incu-

of unlabelled

(2.5; 5; 10; 20; 40 mM) plus 0.2 ,&i

or [U-‘4C]-lactic

substrates,

acid. After

the incubation

addition

medium

of

pH was

determined. EXPERIMENT3:EFFECTOFDlFFERENTGLUCOSECONCEN-

MATERIALS Animals Male

AND METHODS

Chasmagnathus

grunulata

crabs

cycle ( 12) were collected

Grande

do Sul, Brazil, where The

the authorities e dos

animals

aquaria

Naturais

were weighed

(16.48

de Meio Ambiente (Licence

of 15%0, temperature

138/91in

of 22°C and light/

experiments

(120 min)

were performed

were divided

into two groups,

humidity

fibers

0.31%;

a carbohydrate-rich carbohydrates

bers 0.30%;

humidity

HP diet.

Protein

food constituents

meat), low-carbohydrate 0.03%; fat 6.71%; ashes 71.01%),

while

diet (HC-rice)

34.56%;

fat 0.45%;

61.33%),

approximately

the

other

diet (protein

ashes

and carbohydrate

were determined

Institute/UFRGS.

one of which

unlabelled

by measuring

by thin

ethyl

alcohol/5.4%

of crabs

being used in the experiments. groups

No variation

was observed

in body weight

during

the experi-

period.

sulfuric

Tissue

samples

were

obtained

(pmoles) experiments

anesthetized

hypothermia. Hepatopancreas were immediately placed in a Petri dish containing cool-incubating

The

by

removed, medium

and cut into two slices of 200 mg (500 pm thick) each. The fractions were then incubated at 25°C with constant shaking in 3 ml of Pantin-crabs buffer (37) pH 7.8 (isosmotic to hemolymph), in the presence of 0.2 ,uCi [U-‘4C]alanine (171.70 mCi/mmol) or [U-‘4C]-lactic acid (172 mCi/mmol) (DuPont, NEN Products, Boston, MA). Incubation was performed in a Dubnoff incubator with an atmosphere of O,:CO, (95:5, v/v).

The

was sepa-

using: n-butanol/95% in water

(75:47.4:27.6,

to glucose,

reagent

counter

values

localized

by

(95% ethyl alcohol/ 18: 1: 1,

or

with a color quench

of gluconeogenic

lactic

showed

acid

activity

converted

was not measured, that

corare

to glucose

according

than

incorporated

5% of that

determination activity

mogenizer.

since preliminary

the “C incorporated

gen, determined

to Thomas

of

into glyco-

et al. (47),

into medium by Migliorini

was less

glucose.

phosphoenolpyruvate

as described

carboxy-

et al. (32),

from HC or HP groups was homogenized

0.25 M sucrose

ride (PMSF) crabs

14C-glucose

per g hepatopancreas.

ice-cold from

of [U-‘“Cl-

“C-glucose.

acid/p-methoxybenzaldehyde

scintillation

as L-alanine

For

by

experiments

was marked, scraped off, and dissolved in toluene(2 : 1 )-PPO-POPOP. Radioactivity was measured in curve.

kinase

Procedure

acid

with the anisaldehyde

hepatopancreas Experimental

acetic

The 14C in glycogen

for 2 weeks before

and the

layer chromatography

concentrated

given

different

the incorporation

(3). The spot corresponding

spraying

to

the

acid into medium

was deproteinized

rated

isocaloric

Both groups of crabs were fed once daily

in both

medium

rection

(5, 10,

and 20 mM

the rate of gluconeogenesis from

or [U-“Cl-lactic

a LKB Wallac

by the Food Technology

(50 g), ad libitum, in the late afternoon

the incubation,

was determined

triton

of glucose

L-alanine. fractions

v/v/v),

were incubated

of [U-‘4C]-alanine

hepatopancreas

fi-

animals

concentrations

plus 0.2 ,&i

Following

0.02%;

contents

with different

15, 20 mM)

v/v/v)

0.35%;

mental

of

-+ 0.69 g) and placed

(HP-bovine carbohydrate

of animals

from 0.22 to

the permission

Renov6veis

was fed a high-protein diet (protein 21.59%;

the

inRio

the year.

The animals

3.34%;

ranged

Brasileiro

of 12L: 12D. The

throughout

consumed

Lagoon,

slices from HP- or HC-fed

alanine

at a salinity

dark cycle

salinity

were used with

of the lnstituto

Recursos

DEVIS). Animals

in stage C of the

in Tramandai

Hepato-

ON THEGLUCONEOGENESISCAPACITY.

pancreas

termolt

34%0 (48).

TRATIONS

1:3 :0.020,

and phenylmethylsulphonyl (w/v/v)

The homogenate

with

a Teflon

was centrifuged

1 g of in fluo-

pestle

ho-

for 10 min at

600 X g, the supernatant fluid obtained was decanted and recentrifuged at 10,000 X g for 10 min. The sediment of this second centrifugation was washed twice and resuspended to the original volume in 0.25 M sucrose to provide the mitochondrial fraction. fuging the supernatant

The cytosol was obtained by centriof the second centrifugation at

100,000 X g for 1 hr. All steps were carried out at 0-4°C. PEPCK activity was determined through the H”CO;oxalacetate exchange reaction. The reactions were stopped by the addition of 5% trichloroacetic acid. After centrifuga-

Gluconeogenesis

1431

in Ctahs Hepatopancreas

500

! 0

450

02

400

E

350

I-

fi z 300’ &y s” 250 3

t

200’~

y

-

150.

z

-9

2

100 50 o-

tip

, 10

0

_I___

__I____.

L-ALANINE 60

30

120

INCUBATION

*

150

~__.L_

J

50

(mM)

+-

HC

~~

40

30

20

HP

TIME (minutes)

FIG. 1. Glucose synthesis from [U-‘4C]palanine at different times in hepatopancreas slices from crabs fed a high-protein (HP) diet. Data are given as mean f SEM. In parentheses, number of animals.

tion,

the solution

quots

were immediately

spectrometer in which

counted

in a LKB Wallac

ITP was omitted

ues obtained

per mg of protein (31),

in the liquid scintillation

scintillation

counter.

were routinely

were subtracted

The values of PEPCK Protein

Blanks

are given

as pmoles

by the

albumin

method

l

of Lowry

et al.

50

as standard.

“

tic

HP

FIG. 2. Effects of different concentrations of L-alanine or lactic-acid on gluconeogenesis activity in hepatopancreas slices from crabs fed a high-protein (HP) or carbohydraterich (HC) diet. Data are given as mean + SEM of six observav tions.

nificantly

Analyses

increase was found centration

tween

pacity

the experimental

were submitted tests

conditions

were analysed

were

carried

tests Social

were performed

by one-way

out using

test. P < 0.05 was taken

to which Duncan’s

as the criterion

with

the Statistical

the two groups ANOVA;

post

multiple

range

of significance. Package

All

for the

Sciences.

in HC crabs.

In hoth

synthesis

groups,

a linear

in hepatopancreas

as it was exposed to rising medium alanine (2.5, 5, 10, 20, 40 mM). The gluconeogenic

in hepatopancreas

teen-fold

incubated

(P < 0.01) higher,

2.5 mM alanine The effects

of graded

with 40 mM was nine-

in hoth groups,

in the incubation

conca-

than that with

medium.

concentrations

of lactic

acid (2.5,

5, 10, 20,40 mM) are shown in Fig. ZB. The rate of glucose synthesis from 14C-lactic acid increased significantly (P < 0.01)

as a function

of the lactic

acid concentration

in the

medium up to 20 mM. However, it reached a plateau between 20 and 40 mM. The rate of gluconeogenesis from

RESULTS

amount

from that

in the rate of glucose

Data comparing the effects of different diets (HP or HC) were analyzed by two-way ANOVA, while differences be-

The

40

30

LATIC ACID (mM)

fractions.

Statistical

hoc

~-I.

1

20

H’“COI

per minute.

using bovine

_

10

from all assay measurements.

activity

was determined

~1

used, and the val-

The enzyme lactate dehydrogenase (LDH) was used as cytoplasmic marker to ensure low cross-contamination hetween

-9”

was gassed for 2 min with CO2 and ali-

of glucose

synthetized

from L-[UJ4C]alanine

in hepatopancreas fractions from crabs fed a high-protein diet increased with incubation time. At 120 min of incubation, it was 44.3% higher than at 30 minutes (Fig. 1). Figure 2A shows the effects of graded concentrations of alanine in the incubation medium on the gluconeogenic capacity c)f the hepatopancreas from crabs fed HC or HP diets. The incorporation of 14C-alanine into glucose in hepatopancreas from crabs fed a HP diet does not differ sig-

“C-lactate from that

in crabs fed HC diet did not differ significantly in HP diet.

Irrespective

of the experimental

gluconeogenesis from 14C-alanine nificantly higher (P < 0.01) than

conditions,

the rate of

in hoth groups was sigthat from “C-lactate.

Table 1 shows the distribution of PEPCK activity between the mitochondrial fraction and cytosol in hepatopancress from crabs fed HP or HC diets. The results indicate that 89.6% (2.68 -+ 0.44 ~molrs H’YXi per mg of protein

1432

G. T. Oliveira and R. S. M. Da Silva

TABLE 1. Phosphoenolpyruvate carboxykinase (PEPCK) activity in hepatopancreas from Chasmagnathusgranulata fed a high-protein or carbohydrate*rich diet

from*4C-alanine was inhibited by 58% (P< 0.01) in HP and by 41% (P< 0.05) in HC diets. No significant difference was found

between

the HC and HP groups.

PEPCK Activity (pmoles H14COS per mg of protein per min) Mitochondrial activity

Grows

2.68 + 0.44 2.73 2 0.65

HP HC

DISCUSSION

Cytosol activity

The biological

0.31 + 0.07 0.50 ? 0.15

crustaceans ability

per min) of PEPCK mals is observed ? 0.07pmoles cytosol

within

in hepatopancreas

the mitochondria,

In the

HC

group,

H14COI per mg of protein

ity in hepatopancreas

Lactate

H14C0,

and

is an exclusively

and serves as a marker of contamination activity ment

The

present

was detected

data within

and this represent

chondrial

only

0.65

show the

cytosolic

gardless

15.5% is

enzyme,

14% of contamination

of the mito-

pellet.

the incubation

medium,

groups was markedly tration

that

synthesis (37%) diet,

in crabs

fed

glucose

the gluconeogenic

was present capacity

The lowest glucose

a significant

14C-alanine

respectively.

When

decreased.

produced

from

diets.

decrease

was 5 mM

a high-protein

in

in both

and

15 mM

activity.

starvation

In contrast

the intrinsic

synthesis

activity,

shows

do not seem

variations periods)

energy

to what

capacity

from alanine

of

or lactic

to be affected

synthesis

be important

vive great environmental

capacity,

re-

for this crab to sur-

(temperature,

in its natural

consumption,

gluconeogenic

salinity,

habitat,

a pro-

since glucose

capacity

gmnulatu

implies

delivered

to the hepatopancreas

that the amount

fed state does not represent cress

glucose

synthesis,

is the

that crustacean

pool about

10 times

in Chasmqnathzls

of exogenous for hexose

tissues contain

acids in the

for hepatopan-

into account

the size of that

amino

formation

an extra burden

taking

established

glucose

carbon

Irrespective

or carbohydrate-rich

At 20 mM of glucose,

study

that

it i> well

a free amino

found

acid

in Inammalian

tissues, and this pool contains several glucogenic amino acids in sufficient quantities to account for the source of the

concen-

in the glucose

(26%)

The present

The high glucose

of the diet, might

hepatopancreas

compart-

Figure 3 illustrates the inhibitory effect of glucose (5, 10, 15, 20 mM) on the gluconeogenic capacity from’4C-alanine in crabs fed HC or HP diets.

vari-

of gluconeo-

main source of energy in crustaceans (8,20). On the other hand, the fact that a high-protein diet does not increase the

21% of the LDH

mitochondrial

PEPCK

for glucose

cess that requires

during the isolation that

the site(s)

(9,25,34,49),

acid, and the PEPCK by different

activ-

per min)

with

hepatopancreas

in the

(2.732

per mg of protein

about

(1,19,29,38,46).

in vertebrates

and natural

dehydrogenase

procedure.

per min)

per min) of PEPCK

is mitochondrial

(0.50 t 0.15 pmoles within the cytosol.

acid, associated occurs

and 10.4% (0.31 84.5%

in crabs

of the different

to the great interspecies

that the Chasmagnathtls grantllata hepatopancreas has capacity for glucose synthesis from 14C-alanine and “C-lactic

from HP ani-

H14COI per mg of protein

fraction.

pmoles

activity

peculiarities

contribute

and some controversy

genesis Values represent mean 5 SEM, N = S-l 1 animals in each case

and ecological

species

the amount

gluconeogenesis

(22 ).

of labelled

was significantly acid.

atoms

of the experimental higher

In crustaceans,

posmotic

glucose than

ent in tissues

(17,18,46).

of the animals, from 14C-alanine

that recovered

it is well

shock diminishes

condition recovered

from “CZ-lactic

established

the amount Considering

that

of amino

the

hy-

acid pres-

that the Chasmagna-

thus granulatu crab lives in an estuary where salinity ranges from 0.22 to 34% (48), the high glucose synthesis capacity from “C&nine in this crab might be explained by this ecological experiments I fA

60 -

participation

26 0

I 0

20

6

-klc

-

26

nP

3. Effects of different concentrations of glucose on gluconeogenesis activity from [W4C]ealanine in hepatopancress slices from crabs fed a high-protein (HP) or carbohydrateerich (HC) diet. Data are given as mean -C SEM of six observations.

FIG.

peculiarity. carried

This

was clearly

out in our laboratory,

of gluconeogenic

pathway

shown which

in previous suggested

in the adaptation

to the hyposmotic stress in Chasmagnathus granulata (10,36). These findings are consistent with the greatest rate of glucose production found by Thabrew et al. (46) in Cur&us maenas gills, when there were incubated in Na+-deticient sea water. Several data show that L-lactate is the almost exclusive end-product of anaerobiosis in Crustacea (15) and that it accumulates during exercises (5,33) and severe hypoxia or anoxia (6,28,52). However, the fate of lactate is much less

Gluconeogenesis

clear. Gluconeogenesis granulata

1433

in Crabs Hepatnpancreas

from lactate

in the present

of other crustacean gills (1,16,19,38,46),

found in Chasmagnathtls

study is also described

species in muscles, hepatopancreas, In this study, however, it appears

Chasmagnathus granuluta hepatopancreas tiated

gluconeogenic

alanine.

capacities

This difference

possesses

for 14C-lactic

probably

results

gesting

that this tissue may use amino

as precursors

for gluconeogenesis

cean hepatopancreas for lactate tive

reprocessing,

when

served

one

considers

in other

possible

that

crabs

the “C-lactic

the lipids

and bicarbonate

posed

as were found

air (41,42),

of this species

or anoxia

pathways

cd energy

tebrates,

crustaceans

anaerobic

glycolysis,

and

ob-

Also,

it is

among

in-

acid pool and

the

rose quickly

crusta-

(40). Survival by utilization

metabolism.

In contrast

utilize

only

one

glucose

of anaerobic of the acid

found

was submitted

in the present

mechanism

to in this habitat

and the more effective may occur

severe

characteristic

its compartmentalization, enolpyruvate

dria, and microsomes

pathway

and the distribution

of phosphn-

among

derived

lum varieb from species

acid

or anoxia.

of the gluconeogenic

carboxykinase

the cytosol,

(26,34).

is found

within

the

The present

mitochondria.

of the blue crab Callinectes

the deep-water investigated

Chacean the

PEPCK

qlinquedens.

activity

However,

the

product

the diet

a change

These

in

findings

hetween

crabs fed HC

shown

of gluconeogenic

in viva (7,14,26,39,43).

to inhibit

glucose In the

pathway

production

poration

of labelled

were also observed

alanine

into

in isolated

glucose.

hepatocytes

in

hepatopancreas

from HC and HP crabs, graded concentrations in the incubation medium inhibited by 41-58%

of glucose the incor-

Similar

results

from starved

that gluconeogenesis

rats

from alanine

in Chasmagnathus gram&a hepatopancreas IS subject to end-point inhibition by glucose, as has been found in verteIn conclusion,

the

or 14C-acid of this

secretion

gluconeogenic

lactic

associated tissue

from

“C-

Fanttlata

with PEPCK

argues in

functioning

ofdigestive

capacity

in the Chasmagnathus

enzymes

not

only

activity,

as a site for the

but also as a site for gluconeo-

genesis. Thts study wus partially supported by grcuu~from Financiadora de Estudos e Projetos (Conuhio FrNEP/LJFRGS no. 66.91 .OSO9.@0), CNPil and FAPERGS.

These

References 1. Aardt, W.J.Van.

2.

study 3.

results

agree with those of Walsh and Henry (51) and Lallier and Walsh (29), who found significant PEPCK activity in the hepatopancreas

(32,34), induce

in this work that show no

capacity

vitro and

reticu-

shows that in C. gram&a hepatopancreas from animals fed high-protein or carbohydrate-rich diets, 84-89% of PEPCK activity

found

has been

is

mitochon-

from the endoplasmic

to species

does not

of the hepatopancreas.

with results

with

zone),

from “C-lactic

hypoxia

a

the animal

(meso-supra-littoral

gluconeogenesis

only during

A major

work may represent

to the challenge

in vertebrates

(glucose)

favor

lactate, predominantly L-lactate (2,6,44). Therefore, differentiated gluconeogenic capacities for “C-lactic adaptive

that gluconeo-

rates in species

(30,45).

on gluconeogenic

hepatopancreas,

inver-

pathway

activity

are consistent

alanine

into

metabolic

in C. gram&a

difference

at high

on recovery

brates.

severe

to other

basic

is fermentation

to what occurs

peak

of glycogen

and “Galanine

that

blood through

is mediated

Contrary

(43). Our results suggest

is a ex-

and reached

proceeds PEPCK

In vertebrates,

alterna-

in other

with this is the observation

composition the PEPCK

glycogen

or HP diets.

organ

Furthermore, Chasmagnathus grant&a crab that faces hypoxia even when

level 60 min after air exposure hypoxia

the amino

is used to replete

from lactate

intramitochondrial

crusta-

potential

(19,29,45).

acid was distributed

including

to atmospheric

concentration

being a suitable

its gluconeogenic

metaholites,

ceans ( 1,16,46). meso-sllpra-littoral

acids, such as alanine

and in fishes

tracellular

en-

in hepatopancreas in other crabs, sug-

(5 1). Moreover,

the muscle

genesis

differen-

acid and 14C-

may not be the most important

lactate

(30). Consistent

and that

from a higher

zyme alanine aminotransferase activity than lactate dehydrogenase as observed

rate at which

for a number

4.

supidus, and in the

only in a subcellular

authors

frac-

tion obtained at 10,000 X g or 6,500 X g. On the other hand, Henry et al. (19) did not f;nd any significant levels of PEPCK activity in the hepatopancreas of three species of

5.

aquatic 01. terrestrial crustaceans. For animals that routinely undergo periods of prolonged anoxia, as the estuarine Chasmagnathus gram&a, the localization (intramitochondrial) and activity of PEPCK may have an influence on the

6

Lactate metah<)lism and glucose patterns in the river crab, Potamonuutes ujumcm. Calman, during anoxia and subsequent recovery. Camp. Bic>chem. Phyhiol. 91 A:2993@4;1988. Aardt, W.; Wolmarans, C. Effects of ,moxia on the hemolymph physiology and lactate accumulations in the freshwater crab, Potamtm warreni. Camp. R~t)chem. Physiol. 88A:h71675;1987. Baker, N.; Huehotter, R.J.; Schot:, M.C. Analysis of ~luc~>se“C in tissues using thin-layer chtomato~raphy. Anal. Ri<)them. 10:227-235;1965. Botto, J.F.; Yrigoyen, H.R. Bioccologla de la comunidad del cangrejal. I-Contrihucion al conocimient~~ hiolu~ic~~ &I cangrcjo de cstuario, ChasmngnaLhus firnn&w DilM (Crustacea: Decapoda: Grapsidae) en la desrmh~~caclura &I rio S&lo, provincia de Buenos Aires. Sem. L~~tinoam. Ecol. Bentonica y Sedimentation de la Plataforma Continental &I Atlantic~~ Sur, Unesco Montevideo; 1979:161-169. Booth, C.E.; McMahon, B.R.; Pin&r, A.W. Oxygen uptake and the potentiating effects of increased hcmolymph lactate on <>xy,qen transpt)rt during exercise in the blue crab C&nectes sapidus. J. Camp. Physiol. 14X:1 I l-121:1982. Bridges, CR.; Brand, A.T. The effect of hypoxta on oxygen consumption and hloud lactate levels of some marine Crustatea. Camp. Biochem. Phyaiol. 65A: 399-409; 1980.

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