Accumulation of citrate in pancreatic islets of obese hyperglycemic mice

BIOCHEMICAL

Vol. 33, No. 5, 1968

ACCUMULATION

OF

OF

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

CITRATE

OBESE

IN

PANCREATIC

HYPERGLYCEMIC

ISLETS

MICE

F. M. Matschinsky, C. R. Rutherford and J. E. Ellerman Department of Pharmacology Washington University Medical School Saint Louis, Missouri 63110

Received November4, In

several

release,

publications

the

element

in

citrate

the

of citrate order

1968

in

to

molecule

secretory islets

further

Langerhans,

dealing

the

a method

has been

process

have

never

study

of

for

with

the

been measured. the

role

Using

this

made in

the

islet

and acinar

obese

hyperglycemic

strain.

The effect

studied

in

creatic

islets

in

the

mice of

vitro.

It

and of

citrate

of obese

islets

of the

fructokinase

of

hyperglycemic

lean

islets

portions

the

is

It

subject

the

islets

have

of

the

animals

of

levels

the

was demonstrated

to inhibition

of

same

was also in

twice

been

pancreas

of islets

citrate

of

in microscopic

animalswere

controls.

levels in

determinations

control

that

in

citrate

on P-fructokinase

was found

actual

Accordingly,

of

method,

lean

the

of citrate

measurement

of insulin

as an essential

However,

was developed. from

mechanism

implicated

(l-3).

samples

cells

the

pan-

as high that

by citrate

as

Pin

vitro.

Methods Frozen-dried obese cribed ately

and lean (4). after

and plasma matically

specimens mice

Blood the

(C 57 BL/6 was collected

pancreas

separated using

of

islets

of Langerhans

J-ob

strain)

from

was taken

the out.

by centrifugation.

a fluorometric

version

855

and acini

were

sampled

as des-

abdominal

cavity

immedi-

The blood Citrate of

from

the

was heparinized was measured

assay

described

enzyby

Vol. 33, No. 5, 1968

BlOCHEMlCAL

Gruber and Moellering deproteinlzing,

(5).

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Plasma was analyzed directly,

by adding 20 1.11to 1 ml of reagent

fOllOWin@; compositlon:

0.1 Tris-HCl

buffer,

10 MM NADH, and 3 ug/ml of crystalline 10 min at 25O, the fluorescence lyase

buffer,

pH 7.5, which contained

albumin.

(Boehringer)

The reaction

tissue

were extracted

extract,

neutralized

citrate

reagent

of citrate with with

HClOc. Of the

N

as for

of citrate

plasma, except

in microscopic

assay for NAD+ (6-8). was covered

rack was added with oil

(7).

which contained

To 0.1 pg of

After

against

lyase. dilute

and the cooling,

0.3 M Tris-HCl

1 mM MgC12, 30 uM NADH, 0.05 % bovine acid,

samples, the

in small volumes under

20 mln at 70' as described

40 ug/ml of citrate dialyzed

about 50 mg of

placed in the well of a Teflon

50 mM ascorbic

the pro-

was reduced to 5 pM and the standards

0.04 ul was added of a reagent pH 7.8,

through

in whole pancreas

was gained by working

tissue

sample heated for

bumin,

blanks and standards

were carried

0.04 ul of 0.05 M NaOH. The droplet

buffer,

10 min and the fluores-

0.5 ml of cold 0.5

and by using a catalytic

frozen-dried

serum

KsCOJ, 50 ul were added to 1 ml of

to 0.1 mM. For the analysis oil

and 10 to 20 ug of

and the assay was performed

sensitivity

After

dehydrogenase.

1 mM EDTA and 0.05% bovine

Appropriate

that the NADH in the reagent necessary

malic

was completed within

(20 1.11of 0.2 to 0.4 mM citrate) For analysis

the

were added In 2 ~1 of 0.05 M Tris-HCl

cence decrease was measured. cedure.

with

pH 7.8, 0.5 m&f MgC12,

was recorded

citrate

without

serum al-

6 pg/ml of malic dehydrogenase (The serum albumin

Tris-HCl,

and

had been thoroughly

pH 8.0 to remove citrate.)

After

15 min at 25O, 0.12 ul of 0.5 N HCl was added and 10 mln

later

the Teflon

determination following

rack was cooled on ice (7).

For the catalytic

of NAD*, to each well was added 10 ~1 of the

reagent

sodium arsenate,

(8)-: 0.1 M phosphate buffer, 10 mM EDTA, 0.02 % bovine 856

pH 7.4, 10 mM

serum albumin,

5 mM

BIOCHEMICAL

Vol. 33, No. 5, 1968

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ammonium-acetate,

3 mM glyceraldehyde-3-P,

acid,

100 ug/ml

0.3 mM ADP,

genase

prepared

from

nium-sulfate) 75 ul (stored

yeast

of

M NazC03

37O.

tarate,

0.6

15 min

at

~1 of

70°.

the

to 200 ul of

HCl buffer,

pH 9.5,

After

30-40

the

fluorescence

blanks

min

Pancreatic

controls,

larger

(about

higher,

all

was performed

in

according

islet

in

the

tissue containing

albumin,

to

following

mercaptoethanol,

effect

in

glycerol.

M hydrazine-

to the

fluorometer. moles

tubes

citrate)

were are

frozen-dried

larger

samples

citrate

levels

and the

cycling

were are step

Glyceraldehyde-P

concentrations used with Glucose

and

Appropriate

animals

of citrate

manner:

dehy-

were reduced

normal

islets

in plasma

to was de-

100 ul

7.5

to

enzyme

frozen-dried

of 50 mM lmidazole-HCl

buffer,

acetate,

mM sodium

1 mM EDTA, 0.13

1 to

on this 2 ucg of

150 mM potassium

5 mM MgC12,

albumin,

obese

lOOO-fold.

was

(9).

and the

was added

each well

serum

tubes.

of those

for

% bovine

lo-fold

dehydrogenase

of about

heated

from

in addition,

Increased

one-fifth

P-fructoklnase studied

were

and glutamic

amplification

termined

the

a-ketoglu-

0.2

the

and therefore

for

reagent:

3 x lo-l2

10 x 75 mm glass

approximately

give

in

from

Since,

rack

was added

islets

0.5 ug).

volumes

drogenase

to

incubated

dehydrogenase

of NADH recorded

along. from

0.02

glycerol.

an amplifi-

and destroy

droplet

of glutamic

(lo-l3

were

and the

at 37O 1 ml of water

and standards

carried

7.1

0.3 mM NAD+,

mM ADP and 30 ug/ml

To achieve

reaction

following

in

ammo-

glyceraldehyde-3-P

samples

of the

the

dehydrogenase

acid.

cycling

The contents

of excess

to neutralize

3 X Hz02 was added

transferred

to

of glutamic

5000-fold,

To stop

glyceraldehyde-P-dehydro-

by centrifuging

was added

approximately

1 hr at

than

(freed

at pH 3), and a-ketoglutaric

cation

0.3

of NAD-free

and 500 ug/ml

of 0.3

5 mM a-ketoglutaric

0.01

arsenate, mM fructose-6-P,

857

% bovine 0.1

pH

serum

mM NAD+, 0.2

was

1 mM

mM ATP,

Vol. 33, No. 5, 1968

BIOCHEMICAL

45 ug/ml of aldolase,

AND BIOPHYSICAL RESEARCH COMNUNlCATlONS

15 lJg/ml of triose

phosphate

45 ug/ml Of glyceraldehyde-P-dehydrogenase. followed

directly

in the fluorometer.

isomerase,

NADH formation Fructose-1,6-P?

and was

served

as

standard. Results With the oil with citrate

well

and Discussion

technique

concentrations

The overall

from lo-l3

moles of citrate.

to tissue

was 87 %. Pyruvate,

Interfere

at 10 uM concentration.

twice

less than + 10 %). Recovery a possible

in the pancreas

as high as in the controls on a dry weight

was less pronouncea

after

Islets

were also elevated

higher

than in acinar

citrate

concentrations

brief

as well

added did not

as for

The citrate

the

acinar

in the acinar

starvation.

tissue

levels

in

in the obese mice but were 20 to 40 %

tissue

In all

4 groups of animals.

in plasma likewise

in obese hyperglycemic

animals.

parable

plasma sugar levels.

Only after

appeared

The

to be somewhat

The fed groups had comovernight

fasting,

were

lower in the lean animals.

The finding the pathologic

of higher animals

major role

attributed

glycolysis

(10-12).

in vitro

of citrate

1). This was true for

basis,

higher

the levels

was acceptable

of fed obese animals were

The difference

basis.

1)

method (B)

source of error,

(Table

pancreas as a whole on a wet weight tissue

moles (Fig.

method (A) was equivalent

The degree of precision

deviation

of citrate

were linear

and of the less sensitive

(standard

Levels

readings

to 3 x lo-'*

blank of the more sensitive

to 3 x 10” 3 moles of citrate to 10-l*

the final

citrate

deserves

special

to the citrate

in pancreatic attention,

molecule

It has been demonstrated

leads to increased

enhanced insulin

levels

release

levels

by glucose

858

of

because of the

in the regulation that

of glucose-6-P (3).

islets

It is,

citrate In

islets

therefore,

added and con-

of

LEVELS

IN

PANCREAS

I AND PLASMA

fasted

Nutritional

10 hrs. Obese

animals

Lean animals

Half of the animals were used for the determination of citrate in ticroscopic samples of islets and acini. The other half were used for plasma and whole pancreas analyses. The average weight of the controls was 22 Q, the average weight of the obese animals was 58 The mear: of results, the standard errors of the means and the number of animals studied are given, 0.1, $p 0.05, cp’< 0.01 (all compared to controls given the sametreatment).

CITRATE

TABLE

.-d

Vol. 33, No. 5, 1968

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Fig. 1. Yield of product in the catalytic assay for citrate. Assay A is that used for 0.1 pg samples of normal islets; Assay B is that used for 0.5 pg samples of islets from obese mice (see Methods). It is seen that the amplification at the cycling step was approximately 4500-fold for Assay A and nearly WOO-fold for Assay B. i

3 C,TRATE.nl~les

x10"

(8)

3-

2. Effect of citrate on Pfructokinase from pancreatic isleQs of obese mice. The amount of tissue in Assay A was 1.57 ~3 and tn Assay B 1.67 pg. TM arrow indicates when citrate was added to Assay B.

Fig.

0

I

I 10

I

I 20

1

I 30

MINUTES

860

BIOCHEMICAL

Vol. 33, No. 5, 1968

01

fi

’

o

0.2

ceivable

’

0.4 CITRATE

that

animals

is,

plasma

levels

indirect

the at

’

0.6 OR ATP,

*

in

part,

possibility

gerhans

can indeed

be inhibited

increased whether

insulin it

diabetes,

glucose-6-P

(10-12). levels

has further can only

be necessary

in

to determine

investigate

so,

supported

of the

for

the

the

effect

in

the

by in

the

obese

secretion.

861

is

the

by further

effect

studies

citrate

of citrate

of

valid

and,

in

Thus,

on the

the

if

so, of

it

accumulation B-cells

Lanenzyme

understanding

studies.

levels

of

as the

interpretation

mice

of

is probably

islets

same manner

for

effect

known increased

vitro

from

this

obese

of P-fructokinase

P-fructokinase

diphosphate the

islets

to inhibition

implications

the

the

If

Whether

be decided

and fructose

to thoroughly insulin

sources

14).

is

show that

1.5

in

be attributed

2, 3) which

other

’

,.Od

Fig. 3. Inhibition of islet Pf'ructokinase by citrate and ATP. With A!lYT at an essentially noninhibitory level (0.19 mM) citrate was varied as shown. Conversely, citrate was held constant at a level of 0.62 mu and ATP was varied. The basic composition of the reagent is given in Methods. The basal activity with 0.19 mM ATP and no citrate was 267 + 30 mmoles of tictose-1,6-P2 Fer kg dry weight per hour (average of 6 samples 2 standard error). Velocityc and velocityi refer respectively to control velocity (0.19 mM ATP, no citrate) and inhibited velocity.

responsible

(13,

(Figs.

from

*

citrate

of insulin

This

’

0.8 mM

elevated

least

and might

by citrate.

n

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

will on

and also rate

of

Vol. 33, No. 5, 1968

These studies the National

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Acknowledgement were supported by research

Institutes

grant

AM-1059 from

of Health.

References 1.) Maske, H., Diabetes 6, 335 (1957). 2.) R-Candela, J. L., "The Structure and Metabolism of Pancreatic ed. S. E. Brolin, B. Hellman, H. Knutson, Macmillan Islets", New York (1964), 349. 3.) Montague, W. and Taylor, K. W., Nature 217, 853 (1968). 4.) Matschinsky, F. M. and Ellerman, J. E., J. Bfol. Chem. 243, 2730

(1968).

5.) Gruber, W. and Moellering, H., Biochem. J. 346, 85 (19661. 6.) Lowry, 0. H., Harvey Lectures 58, 1 (1963). 7.) Matschinsky, F. M., Passonneau, J. V., and Lowry, 0. H., J. Histochem. Cytochem. 26, 29 (1968). 8.) Matschinsky, F. M., Rutherford, C., and Guerra, L., Proc. 2nd. Internat. Congr. Histochem. Cytochem., New York (1968). 9.) Lowry, 0, H., Passonneau, J. V., Hasselberger, F. X., and Schulz, D. W., J. Biol. Chem. 239, 1.8 (1964). 10.) Passonneau, J. V. and Lowry* 0. H., Biochem. Biophys. Res. Communs. 23, 372 (1963). 11.) Parmeggiani, A. and Bowman, R. H., Biochem. Biophys. Res. Communs. 22, 268 (1963). 12.) Garland, P. B., Randle, P. J., and Newsholme, E. A., Nature London 299, 169 (1963). 13.) Stauffacher, W., Lambert, A. E., Vecchio, D., and Renold, A.E. Diabetologica 3, 230 (1967). 14.) Westman, S., Diabetologica 4, 141 (1968).

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