Studies on biosynthesis of insulin by pancreas slices

BIOCHEMICAL

Vol. 13, No. 3, 1963

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

STUDIES ON BIOSYNTHESIS OF INSULIN BY PANCREAS SLICES* S. R. Wagle Department of Pharmacology Indiana University School of Medicine Indianapolis, Indiana

Received

August

25, 1963

The study great

of the

importance

from the point

mechanisms involved eteology

in the

of diabetes

was made difficult producing

beta

The presence well

because

lack

of the

relative

insulin

problem.

contributed

The development

labeled

insulin

by incubating

pancreas

insulin

serum.

The insulin

reduced

In this pancreatic

communication, tissues

cells

et al. --

in pancreas. tissue

complex

*Supported by United States No. A-04790 and c-06276.

(Farber

diet

may be pre-

ethionine

Public

175

may be

which

and Popper

on insulin fed

anti-

and Forsham 1960).

an ethionine

studies

of

with

enzymes in pancreas

tissue

serum

quantit,ies

extracts

(Grodsky

from rats

as

1960) has made

small

antibody

by feeding

of the ascinar

amounts of insulin

of anti-insulin

of

of proteolytic

study

to the difficulty

assay and isolation

in atrophy

1955 and Armin

sodium sulfite

and the

assay method to measure

the

greatly

this

enzymes in acinar

possible

The presence

sugar

past,

to acinar

and Coval

with

of

of the homeostatic

small

(Moloney

cipitated

per -se is

of blood In the

of an accurate

synthesized

of this

of view

mellitus.

cells

of insulin

control

of proteoletic

as the

newly

synthesis

Health

synthesis

results 1950). by

and normal

Service

diet

Grant

Vol. 13, No. 3, 1963

BIOCHEMICAL

are reported.

Male

fed a normal

chow diet

ethionine

(1 g/kg

were killed.

amino acids

and pooled with

an aliquot

assayed

was then without

incubated normal

The insulin

extract

for

pig

fat

of 0.15

labeled

of pancreas insulin the

M KC1 (pH 2.0),

(pH 9.4)

activity

The acid

with

in Table

biological

activity

of glucose-l-C14

I.

--in vitro

an acid

extract

alcohol

also

to co2 by rat

for

The (Table

These results

is precipitated

alcohol

with.

insulin-like

incubated

into

and serum.

are given

slices

and.

set of aliquots

and assayed

pad method for

amino acids

and this

serum.

oxidation

pancreas

and

was then precipitated

rat

extract

distilled

Another

dicate

L-

and the

running

and. Forsham 1960)

for

gms. of

The in-

was freeze-dried

buffer

a

of acidified

(1960)

against

by Renold -et a1.(1960).

Cl4 from

moleculel.

serum and anti-insulin

complex

assayed

three

Cl4 labeled

the addition

as reported that

of

insulin

in glycine

%pid.idymal

Two to

radioactivity.

The results

rat

from

fraction

(Grodsky

was also

removed

in 2.0 ml.

antibody

radioactivity.

and pancreas

hours

guinea

sodium sulfite

The rats

of Pettinga

The undializable

with

weeks.

a mixture

with

supplemented

two to three

in the

dissolved

diet

together.

twenty-four

residue

by the

for

as present

for

water. the

diet)

by the procedure

dialized

250 gms. were

weighing

and normal

was terminated

ethanol

rats

albino

were incubated

cubation

RESEARCH COMMUNICATIONS

by decapitation

number of rats slices

AND BIOPHYSICAL

with

II) activity

inincorporate extract anti-

stimulates. epididymal

1 (0.25) umoles of each of the following amino acids were added per flask: Alanine, Arginine, ASpartic Acid, Cystine, Glutamic Acid., Glutamine, Glycine, Histiding, Isoleucine, Leucine, Lysine, Phenylalanine, Proline, Serine, Tryosine, and Valine) 176

BlOC~EM~CAt

Vol. 13, No. 3, 1963

AND BIOFHYSICAL

Table

RESEARCH CO~UN~CATIONS

I

INCORPORATION OF Cl4 AMINO ACIDS INTO ACID ALCOHOL EXTRACT AND BINDING OF THIS RADIOACTIVITY Incorporation of '214 A.A.* cw/P*

Total Activity Precipitated without serum present

Condition Normal

wmlg 5540 2 530""s

Ethionine Fed 6890 k 480 * **

***

BY ANTI-INSULIN

SERUM

Total Activity Precipitated with N.S.

Total Activity Precipitated with A.I.S.

cpm/g

cpm/g

240 + 20

1430 f 120

4120 f: 210

260 + 40

1360 + 130

4200 1- lgo

Following abbreviations are used: A.A., amino acids; N-S., normal serum; A.I.S., anti-insulin serum. 2-3 gm of pancreas slices were incubated in 6 ml. of Ringer-b-carbonate medium for ni ety minutes with 2 2.5 x 10 6 cpm of a mixture of Clr: amino acids. A11 activities are expressed per gm of original pancreas. Each figure is an average of six values with S.E. as indicated.

Table OXIDATION Extract

OF GLUCOSE-l-C14

Added

None Normal Serum Acid ethanol extract +Normal Serum Acid ethanol extract +Anti-Insulin Serum Insulin 300 microunits Insulin 900 microunits

II

TO CO2 BY RAT EPIDIDYMAL

No * of Obs.

Activity in ~140~ cpm/gm Wet Tissue 2930 2600

7 7 3 3

FAT PADS*

2 150* 2 220

8700 r 330 3150 f 500 10,500 f 950 20,200 f 1860

*Approximately 500 me; of epididymal fat pad tissue was incubated in 6 ml. of Ringer-bicarbonate medium with glucose-l-Cl4 (2 - 2, 5 x 105 C.P.M.). Normal or antiinsulin serum was added with or without an aliquot of to 25 mg of dialyzed acid alcohol extract quivalent incubated pancreas slices. C3 02 was isolated and counted as BaC03.

Vol. 13, No. 3, 1963

fat

BIOCHEMICAL

indicating

pads,

Furthermore, extract

the

was lost

This provides being

the presence insulin-like

of

Ethionine

that

feeding

increase

Cl4 incorporation

into

cipitated

by anti-insulin

serum.

activity.

of the

with

evidence

RESEARCH COMMUNICATIONS

insulin-like

activity

upon incubation

additional

measured.

AND BIOPHYSICAL

did

acid

alcohol

anti-insulin insulin not

serum. activity

was

significantly

the protein

fraction

pre-

REFERENCES Armin,J., 153:

Grant,R.T. 131,

Farber,E.

and. Weight,P.H.:

J.Physiol.

(London)

1960.

and Popper,H.:

Proc.Soc.Exp.Biol.Med.

-74:

838,

1950.

Grodsky,G.M.

and Forsham,E.H.:

J.Clin.Invest.

-39:

1960.

Maloney,P.J.

and Coval,M.:

Pettinga,C.W.:

Biochem.

Bi0chem.J. Preparations,

2: 6:

179, 28,

1070,

1955.

1960.

Renold,A.E., Martin,D.B., Dagenais,Y.M., Steinke,J., Nickerson,R.J., and Shep6,M.C.: J.Clin.Invest 3: 1487,

1960.

178