Studies on the effects of insulin and acetylcholine on activation of glycogen synthase and on glycogenesis in hepatocytes isolated from normal fed rats

Vol.61,No.1,1974

BIOCHEMICAL AI\ID BIOPHYSICAL RESEARCH COMMUNICATIONS

STUDIES ON THE EFFECTS OF INSULIN AND ACETYLCHOLINE ON ACTIVATION OF GLYCOGENSYNTHASE AND ON GLYCOGENESIS IN HEPATOCYTES ISOLATED FROM NORMAL FED RATS* J.O. Akpan, R. Gardner and S.R. Wagle Department of Pharmacology Indiana University School of Medicine Indianapolis, Indiana 46202 USA

Received

September

16,1974

SUMMARY: The addition of insulin (4.0 x lo-l1 M) or acetylcholine (1O-6 M) to isolated hepatocytes stimulated glycogen accumulation and this stimulation was more pronounced when the medium glucose was raised from 50 to 300 mg percent. Studies with [14C]-glucose showed a two-fold stimulation in glycogen s nthesis by the addition of insulin (4.0 x lo-11 M) or acetylcholine (1O-6 M1 . A sixteen percent increase in the activity of glycogen synthase was observed in cells incubated for 10 minutes with insulin (4.0 x lo-11 M) or acetylcholine (1O-6 M), whereas at one hour incubation a 40 percent increase in activity was observed with the same concentration of insulin or acetylcholine. The effects of insulin and acetylcholine were not additive. It is well absorptive

known that

state.

glycogen

The glycogen

synthase

the active

which

form (I).

"0" to the "I"

insulin

synthesis

exists

of this

still

not clear.

insulin isolated

helped to maintain hepatocytes

comunication of glycogen

better

as studied

we present synthase

active

observed cellular

by electron

evidence

in isolated

(1, 2).

However,

structure

the exact

addition

is of

and more glycogen

microscopy

with

of the

in liver

that --in vitro

for net glycogen hepatocytes

in post

form (D) and

enzyme by the conversion

of the enzyme transformation

We have previously

synthesis

about by the enzyme

the less

about by insulin

mechanism of hormonal regulation

in glycogen

is brought

in two forms:

Activation

form is brought

is involved

(3).

In this

synthesis insulin

in

and activation and acetylcholine.

MATERIALS AND METHODS

liver

Male fed Cox rats

(160-200

parenchymal

were isolated -

cells

*Supported in part by a grant Foundation.

g) were used for all by collagenase

from Eli Lilly 222

@ 1974 by Academic Press, Inc. All rights of reproduction in any form resewed. Copyright

these studies. in Vitro -_I

Rat

Perfusion

and Company and the Human Growth

Vol.61,No.1,1974

technique of cells

as described were

containing trations

previously

incubated

(50-300

were

placed

rpm in an International used for

cellular

Good --et al.

by the

as umoles

glucose/g.

centrifuge

to glucose

which

as described

previously

(7).

incorporated

into

were

suspended

(25 mM) buffer

and sonicated

cell

disruptor

(Model

were

centrifuged

poration

for

computed glycogen

rpm for

of radioactive

glucose

hydrolysis

osazone

synthase After

derivative

assay were

incubation,

the pellet

at 50 volts

with

vial was re-

a sonifier

Inc.).

The samples

in a refrigerated

glycogen glycogen

expressed

EDTA (1 mM), P-mercaptoethanol

20 minutes

into

is

as Cl%]-glucose

was decanted,

seconds

was used to determine

of

and assayed

after

as phenyl

above.

(50 t&l), five

content

was assayed

have been

was

method

hydrolyzed

Glycogen

for

pellet

precipitation

x

at 2000

The resulting

W185 D, Heat Systems-Ultrasonics,

at 2000

the supernatant

Tris

and centrifuged

isolated

the supernatant

ice-cold

the

was washed,

as described

concen-

At the end of incubation,

in glycogen

Kesults

various

(4.0

(6).

was further

(4)

of insulin

tubes

Hepatocytes

centrifuged,

and with

5 minutes.

method

and incubated

in 1 ml of

mixture

glycogen

glycogenlg.

mg of wet weight

25 mM NaHC03 buffer

by the glycogen

oxidase

of glycogen

contents

for

assay

55-75

and in the presence

in iced

Radioactivity

prepared

acids

M) at 37°C.

Centrifuge

glucose

Ringer

(1O-6

The precipitated

as glucose

similarly

Umbreit

mg percent)

glycogen

(5).

Approximately

and 5 mM amino

M) and acetylcholine contents

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

(4).

in 3 ml of

5 n+l lactate of glucose

10'" vial

BIOCHEMICAL

synthase from

centrifuge

activity

and

by the

UDP-El%]-glucose

incor-

(8).

RESULTS AND DISCUSSIONS Results

on glycogen

in the presence can be seen from (low6

With

of insulin this

M) stimulated

in glycogen the

table glycogen

synthesis

increase

synthesis

various

and acetylcholine that

both

concentrations

are

insulin

synthesis

was observed

in glucose

with

concentration

(4.0

223

x 10-l'

100-300

and

It

M) and acetylcholine

hepatocytes.

50 mg percent from

in Table I.

surmnarized

in isolated

with

of glucose

No net

change

glucose in the medium. mg percent

a signifi-

Vol. 61, No. 1,1974

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

224

Vol.61,No.

1,1974

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

cant increase in glycogen synthesis was observed in the presence of insulin The effects

and acetylcholine. no additive

effect

of insulin

were similar

and

was observed when cells were incubated with both insulin

and acetylcholine.

The results on the incorporation

glycogen are summarized in Table II. both insulin

and acetylcholine

and acetylcholine

glycogen by two-fold.

of [14C]-gluocse into

It can be seen from this table that

stimulated the incorporation

A two-fold

stimulation

of [14C]-glucose into

in the incorporation

glucose into glycogen was observed with both insulin

of [14C]-

and acetylcholine

when

50 mg percent glucose was used, although at this concentration,

no net increase

in glycogen synthesis was observed (Table I).

in the incor-

This stimulation

poration of C1'+C]-glucose into glycogen was observed at all studied, although stimulation

of isotope incorporation

higher when lower concentrations that insulin

is not involved

glucose concentrations

was comparatively

of glucose were used. These results suggest

in glucose transport

but is involved in glycogen

synthesis.

Wehave reported in our previous studies (3) that in the absence

of insulin,

800 mg percent glucose was needed to maintain initial

levels.

In the present studies initial

20 Munits/

glycogen

glycogen levels were maintained with

ml (4.0 x 10D1l M) of Insulin,

and with only 200-300 mg percent

glucose in the medium. Conditions under which net glycogen synthesis was observed in these studies with isolated hepatocytes in the presence of insulin are similar

to normal -I_ in vivo post absorptive state.

Studies on the activation

of glycogen synthase are summarized in Table III.

It can be seen from this table that insulin synthase activity

(4.0 x 10-i'

in 10 minutes by sixteen percent.

one hour with insulin

(4.0 x lo-l1 M) showed a further

M) stimulated glycogen

Continued incubation for increase in activity

of glycogen synthase by forty

percent.

showing the effect

on net glycogen synthesis and activation

of insulin

This is the first

report clearly

glycogen synthase in isolated hepatocytes at the physiological of insulin

(4.0 x lo-l1

acetylcholine,

M).

concentration

Although similar

concentration

results are observed with

required is quite high (1O-6 M).

225

of

Miller

and

(6)

17,262 + 1,670

(6)

16,773 + 1,652

(6)

(6)

27,921 2 2,416

(6)

26,268 2 2,319

(6)

24,840 f. 2,431

(4)

55,935 + 6,420

(4)

55,156 54,580

(4)

46,404 5 3,690

(4)

(6)

(‘3 17,801 2 1,591

30,742 2 3,510

12,308 + 2,249

(200 mg%)

9,627 21,752

(100 mg%)

(10)

60,489 2 5,080

(10)

60,348 + 4,590

(10)

60,658 + 5,580

(10)

40,982 + 4,930

(300 mg%)

Isolated hepatocytes had initial *Hepatocytes were prepared as described in Materials and Methods (4). Approximately 55-75 mg of cells were glycogen levels in the range of 150 + 23 pmoles glucose/g/hr. incubated for one hour in 3 ml of Umlkeit Ringer bicarbonate buffer containing 5 mM lactate and 7 mM amino acids mixture and with various concentrations of glucose containing approximately 1 uc of 4CValues shown above are (CPM/g) mean values + glucose ( er 100 mg) and with insulin and acetylcholine. SEM of (N P observations.

ml) + Acetyl(10m6 M)

uunits/3 choline

(20

+ Insulin

M)

+ Acetylcholine

Control

(lO-6

Control

ml)

+ Insulin

(20 punits/

Control

Control

(50 w%)

INCORPORATION INTO GLYCOGEN

CONCENTRATION OF GLUCOSE IN THE MEDIUM

FROM NORMAL FED RATS* AS MEASURED BY "C-GLUCOSE

EFFECTS OF INSULIN AND ACETYLCHOLINE ON GLYCOGEN SYNTHESXS IN ISOLATED HEPATOCYTES

TABLE II

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 61,No.1,1974

TABLE III EFFECTS OF INSULIN AND ACETYLCHOLINE ON GLYCOGENSYNTHASE ACTIVITY IN THE PRESENCE OF 300 MG PERCENT OF GLUCOSE IN ISOLATED HEPATOCYTESFROM NORMAL FED RATS*

---I__--

INCUBATION TIME ---_I*

--.I_

Control

5.9 + 0.6

6.8 + 0.3

(4)

(4)

6.9 + 0.8

9.2 + 0.9

(4)

(4)

6.8 + 0.7

9.8 21.1

(4)

(4)

7.1 + 0.85

9.0 + 0.75

(4)

(4)

--II_--* Control

+ Insulin

(20 punits/

ml)

--CI-CII----cI-Control

+ Acetylcholine

(10N6 M) .-cr_-

Control

+ Insulin

Acetylcholine ----

(20 punits/

INCUBATION TIME (60 mfn)

(10 min)

ml) +

(10s6 M) .-

*Hepatocytes were prepared as described in Materials and Methods (4). Isolated hepatocytes had initial glycogen levels in the range of 150 + 23 pmoles glucose/ g. Approximately 55-75 mg of cells were incubated for one h&r in 3 ml of Umbreft Ringer bicarbonate buffer containing 5 mM lactate and 5 t#M amino acids mixture and 300 mg% glucose and with Insulin and acetylcholine. Values were expressed as mean + SEM of-(N) observations percent glycogen synthetase I increase over glycogen synthetase 0.

Larner

(1) have shown stimulation

in the isolated

this

liver

and have observed

only.

However,

in the present

we were able to observe stimulation

in perfused

of glycogen

liver

(1).

insulin

being absent

clearly

shows that

and not glucose

synthase

perfused

for 6-15 minutes cytes,

of glycogen

maximal effects synthase

activity

that this studies

glycogen

transport

synthesis is mediated

cell

was maximal

isolated

hepato-

than that observed

types which may destroy The data presented

and activation

227

by insulin

for one hour and

is far greater

preparation.

by insulin

effect

with

of insulin

This may be due to other in our hepatocytes

activation

of glycogen

and perhaps

here

synthase

by acetylcholine.

Vol.61,No.1,1974

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

The control versial

of glycogen

due to the lack of direct

(10) and --in vitro

(11) negative

those previously (1) demonstrate the activation

reported that

insulin

agreement with stimulation

as well

by a decrease the present

studies.

--in vivo

presented

(3) and also by Larner

directly

in glycogen

and several

here and et --a al

synthesis

through

mechanisms for this

in the activity

activation

glycogen

stimulates to consider

It would be worthwhile

synthase

These observations

(14, 15),

synthase

in the are in

study,

and acetylcholine

as observed

(12,

in the present

been able to show that

glycogen synthase

in isolated

insulin

hepatocytes, drugs - para-

the exact mechanism of the hormonal regulation

which

might be implicated

to investigate

mechanisms which may include

synthase

the employment of cholinergic

to investigate

glycogen

of glycogen

of both insulin

Since we have, in this

it may be of interest

of parasympathetic

It is known that --in vlvo parasympathetic

studies.

stimulate

as acetylcholine

of hepatic

laboratory

in blood glucose.

causes the release

sympathomimetics,

and due to several

(12, 13) have shown that stimulation

13) which may in turn --in -vitro

9) has remained contro-

However the results

functions

caused a marked increase followed

evidence

reports.

synthase

(l-3,

(1).

Shimazu --* et al nerves

by insulin

in vitro -_I

from this

of glycogen

have been proposed

liver

synthesis

a role for

if this cyclic

activatien

in the receptor is brought

AMP or cyclic

proteins.

about by

GMP.

Acknowledgment We are grateful and their

expert

to Mrs. D. Hutton and Mrs. V. Phelps for the preparation technical

of cells

assistance. REFERENCES

1. :: 4. 5. ;:

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8. 9. 1':: :'3* 14: 15.

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AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Schlender, K.K. and Larner, J.: Annals of Biochem. 25: Thomas, J.A., 486, 1968. Kreutner, W. and Goldberg, N.B.: Proc. Nat. Acad. Sci. (US) 58:1515, 1967. DeWulf, H. and Hers, H.G.: Eur. J. Biochem. 6:558, 1968. Glinsman, W., Pauk, G. and Hern, E.: Biochem. Biophys. Res. Comn. 39:774, 1970. Shimatu, T., Fukuda, A. and Barr, T.: Nature 210:1178, 1966. Shimazu, T.: Science 156:1256, 1967. Frohman, L.A., Edzini, E.Z. and Javid, R.: Diabetes 16:443, 1967. Kanetas, A., Kosaka, K. and Nakas, K.: Endocrinology 80:530, 1967.

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