Vol. 64, No. 1,1975

BIOCHEMICAL

STUDIES ON THE DIFFERENTIAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

RESPONSE TO INSULIN

ON THE STIMULATION

OF

AMINO ACID INCORPORATION INTO PROTEIN IN ISOLATED HEPATOCYTES CONTAINING DIFFERENT LEVELS OF GLYCOGEN*

S.R.

Wagle and L. Sampson**

Department Indiana

University

Indianapolis, Received

March

of Pharmacology School Indiana

of Medicine 46202

(USA)

7,1975 SUMMARY

Effect of insulin on amino acid incorporation into protein by isolated rat liver hepa cytes was studied. A two to three-fold increase in the incorand U- I%-Phenylalanine poration of U- 18 C-Leucine into protein by insulin (100 UUnits) was observed in isolated hepatocytes containing high glycogen. This effect was abolished by the addition of glucagon (3 x lo- M). No stimulation in amino acid incorporation by insulin was observed when isolated hepatocytes contained low or no glycogen. Electron micrographs of incubated cells show that in the presence of insulin more normal parallel strands of polyribosomes are maintained as compared to control cell preparation. Insulin of protein diabetic

is

generally

synthesis animals

measured

(1,

However,

known.

In the present and stimulation

in isolated

hepatocytes.

*Supported Foundation.

by a grant

**Present

address:

Eli

as a protein-anabolic

in several

and can be restored

insulin

levels

2).

accepted

to normal

the primary

studies

ways is

site

we report

hormone.

subnormal

incorporation

from

Lilly

and Co. and the

Lilly

Copyright Q I9 75 by Academic Press, Inc. All rights of reproduction in any form reserved.

defect

an interrelationship

acid

and Company, 72

Indianapolis,

into

of

of animals

metabolic

of amino

Eli

in tissues

by treatment

of this

The rate

is

between protein

not glycogen

by insulin

Human Growth Indiana.

with

Hormone

BIOCHEMICAL

Vol. 64, No. 1, 1975

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

MATERIAL AND METHODS Male rats weighing 180-200 g, fed or fasted (6-8 hrs; 16-18 hrs), All rats were maintained on Purina were used in all studies reported here. Laboratory Chow and tap water fed ad libitum. The rats received their food It was previously observed (3) that in a dish that was placed on the floor. liver glycogen was significantly increased in animals that had easy access to the food as compared to those rats that had to obtain their food from suspended wire baskets. Rat liver cells were isolated by collagenase in vitro perfusion technique as described previously (4). Approximately E-70 mg cells were incubated in 3 ml of Umbreit Ringer, 25 mM NaHC03 buffer (5) containing 7 mM glucose, 5 M lactate and 5 mM amino acids (containing 0.5 pc of u- C-leucine or U- T4 C- phenylalanine) and with various concentrations of hormones at 37'C and at 90 oscillations per minute for 1-3 hrs. The vials were gassed with 95% 02 and 5% CO2 for 5 min. at time zero and after each 1 hr of incubation. Insulin, 50 UUnits, was added on timg zero and 25 PUnits was added at the end of one and two hours. Glucagon (lo- M) was added at time zero, one and two hrs. At the end of the incubation period the reaction was stopped by the addition of trichloracetic acid and radioactivity in the protein was assayed as described previously (6,7). Cellular glycogen was precipitated by the method of Good --et al. (8), hydrolyzed and assayed by glucose oxidase method (9). The ultrastructure studies were carried out by thin section electron microscopy on the Sieman IA polarizing electron microscope. Cells after incubation (1 hr) were fixed overnight at O°C with 2% glutaraldehyde in 0.1 M Na phosphate (pH 7.3) buffer. The cells were rinsed twice with 0.1 M Na phosphate buffer and post-fixed with 2% osmium tetroxide in 0.05 m Na phosphate buffer for one hour at O'C. They were rinsed again in 0.05 M Na phosphate buffer and dehydrated in a graded ethanol series to 100% ethanol. Infiltration was completed with propylene oxide and samples were embedded in Epon-812 resin. Thin sections were cut with LKB ultratome. They were stained with 2% uranyl acetate in absolute ethanol and post-stained with Reynolds lead citrate (10).

i5

RESULTS AND DISCUSSION The incorporation by isolated leucine the

hepatocytes

is

presence

incorporation

insulin

(100

of these

studied,

reaching

Addition

of glucagon amino

into

protein

were

incubated

(3 x 10-6M).

increased

of 7.5 mM glucose,

with

amino

showed

acids

into

with

hepatocytes

a progressive

effect

was abolished

by added

73

in the

incubation

periods

incorporation

amino

acid

by glucagon UUnits)

insulin

in

mixture.

by the end of three on the

(100

acids

time

increase

at various

three-fold

of insulin

protein of labeled

incubation

and 5 mM amino

had no effect

into

The incorporation

Insulin-stimulated

combinations

No stimulatory

I.

protein

of almost

protein.

by isolated with

5 mM lactate

(3 x 10V6M) into

in Table proportionately

UUnits)

an increase

acids

and U-14C-phenylalanine

summarized

and phenylalanine

Treatment

these

of U-l4 C-leucine

hours.

of incorporation when cells

and glucagon

was observed

in isolated

2

1320 + 175

1056 5170

925 +110

1280 + 180

92

1560 + 160

810 +

2080 -+ 190

1120 + 150

1 HR

u-14c

PHENYLALANINE

2130 + 240

1960 5 210

4260 + 410

1880 5 200

LEUCINE

uJ4c2 HR

1680 5160

1520 + 130

2975 + 330

1380 5110

PHENYLALANINE

u?c-

HEPATOCYTES FROM FED RATS* (dpm/mg

3650 +

3480 +

420

400

8800 21680

340

&‘4C-

2260 + 280

2100 + 240

4650 + 360

1760 +190

PHENYLALANINE 3 HR

(J?cLEUCINE

3260 +

protein)

AND U-14-PHENYLALANINE

Isolated hepatocytes had initial glycogen levels in Hepatocytes were prepared as described previously (4). Approximately 55-75 mg of cells were incubated for l-3 hours in 3 ml the range of 185 + 35 pmoles glucose/g. buffer (5) containing 7.5 mM glucose, 5 mM lactate and 5 mM amino acids mixture a,,'d"~~~e,a~ns6n,g"~5b~~a~~o~~~~C-amino acid. Values are (dpm/mg protein) mean value + SEM of 6 observations.

CONTROL + INSULIN (100 PUnits) + GLUCAGON (3 x lD6M)

CONTROL + GLUCAGON (3 x 10-6M)

CONTROL f INSULIN (100 UUnits)

CONTROL

PERIOD OF INCUBATION

LEUCINE

&-

I

AND GLUCAGON ON THE INCORPORATION OF U-14C-LEUCINE

INTO PROTEIN IN ISOLATED

EFFECT OF INSULIN

TABLE

i?l v) r k 0

g

5 9 d ii

:

E

g

2:

4o*f_ 20

NONE**

NONE**

CONTROL + INSULIN (100 UUnits)

CONTROL

CONTROL + INSULIN (100 nUnits)

680 +

720 + 78

90

1180 5 160

960 2 140

1 HR

uJ4c-

80

580 +

620 + 70

85

970 + 100

730 +

PHENYLANANINE

uJ4c-

+ 190 2210 +180

2130

3880 + 210

uJ4c-

1420 5160

1380 tl50

1976 + 140

1520 + 130

PHENYLANANINE 3 HRS

2620 + 180

LEUCINE

Hepatocytes were prepared from 6-8* hr or 16-18** hr fasted rats as described previously (4). Approximately 55-75 mg of cells were incubated for l-3 hrs in 3 ml of Umbreit Ringer Bicarborate buffer (5) containing 7.5 mM glucose, 5 mM lactate, and 5 mM amino acids and containing 0.5 PC of U- C-amino acid. Values are (dpm/mg protein) mean values + SEM of 4 observations.

49t20

GLYCOGEN LEVELS pm01 glucose/g

CONTROL

PERIOD OF INCUBATION

LEUCINE

protein)

IN ISOLATED

HRS) GLYCOGEN (dpm/mg

AND U-14C-PHENYLALANINE

HRS) AND NO (FASTED 16-18

&‘4C-

LOW (FASTED 6-8

ON THE INCORPORATION OF U-14C-LEUCINE

HEPATOCYTES CONTAINING

EFFECT OF INSULIN

TABLE II

H

iii z-i >

ii 3 ii z

G 2t

3

$: .%

Vol. 64, No. 1,1975

Figure

1-A:

hepatocytes is

show a small This

ficant

(control)

incubated

only

Studies

fasted

for

rats

Hepatocytes

in the incorporation

in agreement in amino

effect

with acids

x mag) of isolated

one hour

obtained

previous

of insulin

on ultrastructure in Figure

into

animals.

on amino

that

from

contain

of incubated I (A-D).

76

fasted acids

high

Cells

rats into

(16-18 protein

where

studies

suggest into

concentration (1 hr) incubated

hrs) (Table

a signi-

was observed

incorporation

cells

I.

was low as

(3)

protein These

acid

in Table

content

observation

incorporation

hepatocytes

as described

of amino

from 24 hr fasted

in hepatocytes

are summarized

our

RESEARCH COMMUNICATIONS

or when glycogen

decrease

obtained

the stimulatory

from

(22,400

II.

is

hepatocytes

ditions

micrograph

AND BIOPHYSICAL

in Table

decrease

observed

Electron

obtained

evidenced

II).

BIOCHEMICAL

in that

protein

is

of glycogen. under with

various insulin

conshowed

VoL64,No.

Figure

high

I-B:

Electron

micrograph

[control described

+ Insulin in Table

intracellular

glycogen,

polyribosomes glucagon there

BICKHEMICAL

I,1975

is

similar

(Fig.

1-B)

(22,400

as compared cells

an increase

in free

ribosomes

and insulin

here

suggest

that

and that

and glucagon insulin this

is

(D).

are treated

involved

synthesis

with with

treated

1-C)

incubated

I.

associated

changes

x mag) of isolated

(50 utlnits)]

and insulin

ultrastructural

synthesis

AND BIOPHYE~CAL RESEARCH COMMUNlCATlONS

more normal

control

(A),

In the absence and a decrease

also

(Fig.

directly

may be mediated

both

hepatocytes 1 hour

strands

or glucagon

(C),

of insulin

(Fig.

in polysome

1-D).

as

parallel

seen in the presence

cells

77

for

and 1-A)

strands

and

of glucagon

The results

in glycogen

of

(Fig.

presented

and protein

by a common messenger

or

Vol.64,

No. 1,1975

Figure

that

1-C:

glycogen

BIOCHEMICAL

Electron (control described

may act

micrograph + glucagon in Table

as the

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

(22,000 x mag) of isolated hepatocytes 10-6 M) incubated for one hour as I.

stabilizing

factor

to maintain

polyribosome

strands. Crane both

and Miller

stimulated

observed

plasma

in our in

isolated

16-40%.

Pain

-et --al rat

have shown

protein

previous

synthesis

to a diabetic

(11)

studies hepatocytes

(14)

resulted

that

synthesis (12,

insulin

and cortisol

by isolated 13) that

and also

activated

have shown previously in reversion

of rough

78

insulin

that

succinate

hepatocytes.

We have

stimulated glycogen

glycogen synthase

administration

endoplasnic

reticulum

by

of insulin to

BIOCHEMICAL

Vol. 64, No. 1,1975

Figure

I-D:

Electron (control incubated

parallel

arrangement

decrease

in the

of the

of smooth

increased.

A decrease

by isolated

hepatocytes

in insulin-deficient

(15).

normal

cells

also

helps

effect

The studies not

to maintain

requires We are

of cells

only

high

grateful

and their

in amino

presented

stimulates normal

parallel

to Mrs. expert

cell

reticulum acid

show that

acid strands

with

a concommitant

and glycogen

incorporation

animals

here amino

intracellular

normal

endoplasmic

was greatly

ported

RESEARCH COMMUNICATIONS

micrograph (22,400 x mag) of isolated hepatocytes t Insulin, 50 uunits, t glucagon, 1 x 10-5 M) for one hour as described in Table I.

characteristic

amount

AND BIOPHYSICAL

into

content protein

has been previously addition

incorporation

of insulin into

of polyribosomes

protein

reto but

and this

glycogen. V. Phelps

technical

and Ms. Ann Schwab

assistance.

79

for

the preparation

Vol. 64, No. 1,1975

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

REFERENCES

:: 3. 4.

W.D. Lotspeich, J. BIol. Chem. 179:175, 1949. D.M. Kipnis and G.F. Cori, J. Biol. Chem. 224:681, 1957. S.R. Wagle, and W.R. Ingebretsen, Jr., Proc. Sot. Expt. Biol. and Med. 147:581, 1974. W.R. Ingebretsen, Jr., and S.R. Wagle, Biochem. Biophys. Res. Comm.

46:403, 1972. 5. 6. 7.

8. 1::

11.

12. 13. 14. 15.

W.W. Umbreit, R.H. Burris and J.F. Stauffer, Monometric Technique P. 155, 1964. S.R. Wagle, Biochem. Biophys. Res. Comm. 59:1366, 1974. D. Monier, S. Santhaman and S.R. Wagle, Biochem. Biophys. Res. Comm. 46:1881, 1972. C.A. Good, H. Kromer and M. Somagi, J. Biol. Chem. 100:485, 1933. S.R. Wagle and J. Ashmore, J. Biol. Chem. 236:2868, 1961. F.S. Reynolds, J. Cell. Biol. 17:208, 1963. L.F. Crane and D.L. Miller, Biochem. Biophys. Res. Comm. 60:1269, 1974. S.R. Wagle, W.R. Ingebretsen, Jr. and L. Sampson, Biochem. Biophys. Res. Comm. 53:937, 1973. J. Akapan, R. Gardner and S.R. Wagle, Biochem. Biophys. Res. Comm. 61:22, 1974. V.M. Pain, J. Lanoix, J.J.M. Bergerson and M.J. Clemens, Biochemica Biophysics Acta 353~487, 1974. W.R. Ingebretsen, M.A. Moxley, D.O. Allen and S.R. Wagle, Biochem. Biophys. Res. Comm. 49:601, 1972.

80

Studies on the differential response to insulin on the stimulation of amino acid incorporation into protein in isolated hepatocytes containing different levels of glycogen.

Vol. 64, No. 1,1975 BIOCHEMICAL STUDIES ON THE DIFFERENTIAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS RESPONSE TO INSULIN ON THE STIMULATION OF A...
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