Interrelationship of insulin and glucagon ratios on carbohydrate metabolism in isolated hepatocytes containing high glycogen

Interrelationship of insulin and glucagon ratios on carbohydrate metabolism in isolated hepatocytes containing high glycogen

BIOCHEMICAL Vol. 67, No. 3, 1975 INTERRELATIONSHIP METABOLISM AND BIOPHYSICAL RESEARCH COMMUNICATIONS OF INSULIN IN ISOLATED AND GLUCAGON RATIOS...

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BIOCHEMICAL

Vol. 67, No. 3, 1975

INTERRELATIONSHIP METABOLISM

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

OF INSULIN

IN ISOLATED

AND GLUCAGON RATIOS

HEPATOCYTES S.R.

Department Indiana

October

HIGH GLYCOGEN+

Wagle of Pharmacology

University

School

Indianapolis, Received

CONTAINING

ON CARBOHYDRATE

of Medicine

Indiana

46202

6,1975 SUMMARY

The effect of physiological concentrations of glucagon and insulin on glycogenolysis was studied in the presence and absence of substrates in isolated hepatocytes containing high In the absence of substrates glucagon stimulated glycogen. glycogenolysis at lo-14M concentration, and addition of 100 punits of insulin partially inhibited glucagon stimulated glycogenolysis (10'14M to lo-11M). However, in the presence of substrates, insulin completely inhibited glucagon stimulated glycogenolysis (lo-l4 M to lO-llM), indicating that molar glucagon and insulin ratios control carbohydrate metabolism in liver. Additional studies showed incorporation of amino acid into protein was linear for only 3 to 4 hr in cells containing low whereas in cells containing high glycogen, incorporaglycogen, tion was linear for 8 to 10 hr. It

has been

glucagon acid

implicated

participates

metabolism

of these

in the

in hepatic

two hormones

removed

from

that

the

acids

are utilized

Unger

determine

extracellular protein

(2)

has postulated

that

may control

carbohydrate

'This

the

effect

investigation

regulation

tissues

for

report

of

insulin

is

supported

of glucose (1).

whether

Relative glucose

with

and amino concentrations

is

added

or

compartment,

and whether

biosynthesis

or gluconeogenesis.

molar

metabolism. glucagon

in combination

glucagon

to insulin

In this

and insulin

amino

ratios

communication

we

on glycogenolysis

at

by USPHS grant

AM 14340.

Vol.67,

No. 3, 1975

physiological tions evidence

BIOCHEMICAL

concentrations

with

isolated to

support

under

hepatocytes this

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

various

and provide

experimental the

first

condidirect

hypothesis.

MATERIALS

AND METHODS

Male, fed Cox rats (160-200 g) were used for all studies. All rats were maintained on Purina Laboratory Chow and tap water fed & libitum. Rats received their food either placed on the floor (for high glycogen levels) or suspended in wire baskets (for low glycogen levels) as it was previously observed that glycogen levels were significantly increased in animals that had easy access to food as compared to rats that Rat had to obtain their food from suspended wire baskets (3). liver cells were isolated by collagenase --in vivo perfusion technique as described previously (4, 5). Cells from two rats were pooled together as all incubations could be carried out with the same cell preparations. Approximalety 60-75 mg of cells were incubated in 3 ml of Umbreit-Ringer 25 mM bicarbonate buffer with various concentrations of hormones and with and without substrates at 37OC and 90 oscillations per min (6). The vials were gassed with 95% 02 and 5% CO2 for 5 min at time zero and after each 2 hr of incubation. At the end of incuba-

Figure 1. Light micrograph (80 x mag) of normal isolated liver cells just prior to incubation. Liver cells are completely dissociated from each other and demonstrate intact cell membranes and not a single broken cell may be seen. They are a?so completely free of broken connective tissue and red blood or Kupffer cells.

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Vol. 67, No. 3, 1975

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

tion the medium was assayed for glucose by the glucose oxidase method (7). Protein was precipitated by the addition of trichloroacetic acid to the entire vial contents and radioactivity into protein was assayed as described previously (8). RESULTS AND DISCUSSION Figure present

1 shows typical

study.

completely

It

can be seen from

dissociated,

figure

that

cell

may be seen

in the

completely

free

from

connective

or Kupffer

cells

a single

They

are

also blood

this

used

cell

not

broken

demonstrate

hepatocytes

distinct

and

and red

isolated

broken

and all

cells

in

the

cells

are

membranes entire

exclude

field. tissue trypan

Figure II; Electron micrograph (16000 x mag) of a normal hepatocyte after isolation. Mitochondria and rough endoplasmic reticulum are normal. Parallel strands of polyribosomes are also present and do not show any presence of vacuoles. Large number of microvilli are also present on the surface of the cell membranes.

1021

Vol. 67, No. 3, 1975

blue.

BIOCHEMICAL

Figure

mediately

II

after

intact

cell

of rough

is the

an electron

micrograph

isolation.

It

membrane,normal

endoplasmic

reticulum

show any vacuoles

tions

have been

that

of both

collagenase

numbers

of microvilli which

physiological cell

is

(5)

tions

we were able

mulated high

observed

Table

I that

amounts

glucagon

of

in

as reported

M to

served

in

cytes

that Addition the

obtain

hepatocytes

when glucagon 10e6M, the

whereas

present

contained of

more

100 uunits

glycogenolysis

under

can be sti-

contained (lo-'H)

of

on glycolow glycogen,

a two-fold

similar

physio-

(10'14M) that

Cl.21 increase

with

cells

were

increased

a three-fold

enprepara-

It

contained

previously

than

Further-

The results

effects

concentrations

studies high

that

con-

normal

concentrations

Less than

was observed

at

I and II.

stimulatory

(12).

such cell

hepatocytes

Much higher

to

previously

low glycogen

isolated

high

in

of cytoplasmic

of glucagon

at

missing

and glucagon.

in Table

addition

of cell

(9-11).

response

insulin

large

responses

are

Using

hormonal

summarized

isolated

glycogenolysis

blocked

cells.

glycogen.

were needed

genolysis

lo-lo

in these

in

hormonal

by using

or depletion

glycogenolysis

surface

microvilli

swelling

the

addition

and hyaluronidase

of both

are

prepara-

In

on the

prepared

granules

concentration

(9-11).

These

to obtain

studies

cell

high

to obtain

collagenase

concentrations

seen from

using

shows

strands

glycogen

in other

present

have been

no mitochondrial

of these

also

may be essential

zymes

in

are

of both

logical

by

concentrations.

centration more

as seen

im-

cell

Parallel

and abundant

prepared

that

of hepatocyte

can be seen that

and hyaluronidase

preparations

RESEARCH COMMUNICATIONS

mitochondria,

and do not

membranes

AND BlOPt-lYSlCAL

containing from

increase conditions

is obin hepato-

glycogen. of

insulin

when the

1022

(4 x 10-llM) glucagon

partially

concentration

was

Vol. 67, No. 3, 1975

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE EFFECTS OF VARIOUS ITS

INHIBITION

CONCENTRATIONS

BY INSULIN

I OF GLUCAGON ON GLYCOGENOLYSIS

IN ISOLATED

HIGH LEVELS OF GLYCOGEN INCUBATED

Hormone

Concentration

None 10m6M Glucagon 10m6M Glucagon 10W7M Glucagon 10s7M Glucagon lO'*M Glucagon lO'*M Glucagon lo-'M Glucagon lo-'M Glucagon 1O'l'M Glucagon 10'loM Glucagon 1O'llt.l Glucagon 10'llM Glucagon lo-l* M Glucagon lO'l*M Glucagon lo-l3 M Glucagon 10-13M Glucagon lO'l4M Glucagon 10'14M Glucagon 10'15M Glucagon lo-l5 M Glucagon

HEPATOCYTES

AND

CONTAINING

IN THE ABSENCE OF SUBSTRATE+

umoles glucose released in the medium per gram per hour

+ 100 PUnits

Insulin

+ 100 uUnits

Insulin

+ 100 PUnits

Insulin

+ 100 MUnits

Insulin

+ 100 PUnits

Insulin

+ 100 PUnits

Insulin

+ 100 PUnits

Insulin

+ 100 PUnits

Insulin

+ 100 PUnits

Insulin

+ 100 DUnits

Insulin

66.2 220.5 206.4 214.3 200.0 195.6 180.0 182.2 167.0 165.3 148.1 145.3 108.0 125.6 89.0 105.5 12.8 83.6 73.6 68.8 66.8

'Approximately 60-75 mg of cells were incubated in Ringer 25 mM bicarbonate buffer containing various of glucagon and insulin for 1 hr. Initial liver were in the range of 340 + 35 umoles glucose per fusing. Isolated liver cells had glycogen in the 32 umoles glucose per gram. Values are expressed released in the medium per gram per hour.

1023

f t ?r k f f t f i f + f f f f + t f f f f

7.3 22.5 20.0 21.0 19.0 18.0 12.0 16.2 12.0 15.0 12.3 13.0 13.0 9.0 9.2 8.6 8.1 9.8 9.5 9.2 8.6

3 ml of Umbreitconcentrations glycogen levels gram before perrange of 276 2 as umoles glucose

Vol. 67, No. 3, 1975

BIOCHEMICAL

AND BIOPHYSICAL

TABLE EFFECT OF VARIOUS ITS

INHIBITION

CONCENTRATIONS

BY INSULIN

II OF GLUCAGON ON GLYCOGENOLYSIS

IN THE ISOLATED

HIGH LEVELS OF GLYCOGEN INCUBATED Hormone

None 10m6M 10W6M 10W7M 10a7M 10'8M 10e8M 1O"M 10mgM 10'loM lo-lo M 10'llM 10-llM lo-l2 M lO'l*M 10'13M 10'13M 1O'l'M 10-14M 10'15M 10-15M

'The conditions mixture also mixture (6).

in the

range

(Table

I),

HEPATOCYTES

IN THE PRESENCE

Concentration

Glucagon Glucagon Glucagon Glucagon Glucagon Glucagon Glucagon Glucagon Glucagon Glucagon Glucagon Glucagon Glucagon Glucagon Glucagon Glucagon Glucagon Glucagon Glucagon Glucagon

RESEARCH COMMUNICATIONS

umoles in the

+ 100 PUnits

Insulin

+ 100 PUnits

Insulin

+ 100 PUnits

Insulin

+ 100 Wnits

Insulin

+ 100 PUnits

Insulin

+ 100 pUnits

Insulin

+ 100 PUnits

Insulin

+ 100 PUnits

Insulin

+ 100 pUnits

Insulin

+ 100 pUnits

Insulin

AND

CONTAINING

OF

SUBSTRATE+

glucose released medium per gram per hour 58.8 206.0 155.0 180.0 142.0 166.0 130.0 148.0 115.0 134.0 102.0 112.7 68.0 101.0 63.2 88.9 65.0 76.2 60.5 60.3 62.2

f 6.2 + 22.0 f 12.0 f 20.0 + 11.0 k 18.0 2 10.0 f 15.0 k 11.0 f 12.0 + 9.0 f 9.8 f 8.0 * 9.0 f 8.0 + 10.2 f 6.0 f 7.0 ?1 6.0 k 7.1 zt 6.8

were as in Table I except that the incubation contained 5 mM lactate and 5 mM of amino acids

of 10 -14M to whereas

at

lo-l1

higher

M in

the

concentrations

1024

absence

of added

of glucagon

substrates it

was

BIOCHEMICAL

Vol.67,No.3,

1975

much less

effective.

II)

However,

such as 5 mM lactate

of 100 uunits at all

but

sults

clearly

of

the

rect

disease

that

in the

ly reported

(3)

increased to those

a ratio

molar

of

ratios

control

of

the

in animals

that

that

have

of substrates

the

insulin

carbohydrate by Unger

(1,

2).

the

first

insulin

in the

from

controls di-

may be in various

We have previous-

have easy access to food

These re-

metabolism

content

to obtain

glycogenolysis

and provide to

(Table

addition

to glucagon

of glucagon

glycogen

mixture,

of glucagon.

metabolism,

that

rats

blocked

concentrations

as suggested

states,

presence

completely

carbohydrate

evidence

important

insulin

highest

in the

RESEARCH COMMUNICATIONS

and 5 mM amino acids

show that

the hepatic

AND BIOPHYSICAL

liver

food

can be

as compared

suspended wire

TABLE III INCORPORATION OF l4 C PHENYLALANINE

INTO PROTEIN BY ISOLATED

HEPATOCYTES* CONTAINING LOW AND HIGH GLYCOGEN

14 Period of Incubation

Incorporated dpm/mg

Low Glycogen**

1 2

530 f

65

a20 +

a9

High

into

Glycogen***

582 +

72

880 2

a6

3

1080 + 120

1205 + 108

4

1405 + 130

1830 2 128

6

1686 + 160

2663 2 150

a

2063 t

180

3580 2 210

2230 5 210

4752 2 320

10 *Approximately bicarbonate lin

C Phenylalanine Protein

60-75 buffer

mg of cells containing

and 5 mM of amino

Glycogen

levels

were

acids

were

incubated

5.5 mM glucose, mixture

130 + 25**

containing

and 220 + 35***

gram of cells.

1025

in 3 ml of Umbrelt-Ringer 5 mM lactate, 0.5

100 uunits

25 mM of insu-

pCi of 14C phenylalanine

pmoles

glucose

as glycogen

(6). per

Vol. 67, No. 3, 1975

These

baskets.

respond in

isolated

present

ported

reported (lo-'M

to

and also

(12,

16-19)

10B6M> of these

responses.

It

glycogen

of glucagon insulin

is in

where

high

(10-14M)

(4 x 10-llM'I)

contrast

high

--in -vivo

to previously

much higher

concentrations

intracellular

metabolic

as

as re-

hormones are needed to obtain

may he that

to maintain

to

This

(13-15).

studies

containing

concentrations

studies

previously

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

hepatocytes

to physiological

the

helps

BIOCHEMICAL

similar

glycogen

characteristics

content of

isolated

cells. Studies

on the

low and high in Table alanine of

III.

It

into

protein

of It

activity

only

was also

fraction.

was being

rapidly

tion

of amino

tive

protein

synthesis

to bacterial

growth

(750

y/3

tics

were not

into protein

since

added

both to the

large

when incubations

more

under

than

that

newly

the

medium.

the

in the

presence con-

incorpora-

50%

condi-

of the in

the

radiosuper-

synthesized

protein

The incorpora-

presumably

represents

hepatocytes

ac-

and is

not

penicillin

and streptomycin

incubation

medium.

of bacteria No bacterial

were carried

out

phenyl-

similar

was present

numbers

6 hr of incubation.

U-14C

In cells

increase

by isolated

ml> were added

end of

served

released

that

mixture.

protein

suggests

into

table

in

summarized

is

was observed

into

acids

acids

that

protein

8 to 10 hr in

a small

observed

This

this

for

and amino

incorporated

natant

the

was linear

lactate

into

hepatocytes

U-l4 C phenylalanine

tions.

incorporation

can be seen from

low glycogen

tion

acid

glycogen-containing

glucose,

taining

amino

If

antibio-

were noted growth

in the

presence

ACKNOWLEDGEMENT to Deborah

Hutton,

1026

at

was ob-

antibiotics.

I am grateful

due

Ann Schwab and Diane

of

BIOCHEMICAL

Vol. 67, No. 3, 1975

Bauchle technical

for

the

preparation

assistance

AND BIOPHYSICAL

of cells

and Linda

Sampson

RESEARCH COMMUNICATIONS

and for for

their electron

expert micro-

graphs. REFERENCES ::

3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

Unger, R.H. (1972) Isr. J. Med. Sci. 8, 252-259. Unger, R.H. (1974) Metabolism 23, 581-589. Wagle, S.R. and Ingebretsen, W.R., Jr. (1974) Proc. Sot. Expt. Biol. and Med. 147, 581-584. Ingebretsen, W.R., Jr. andxgle, S.R. (1972) Biochem. Biophys. Res. Comm. 47, 403-410. Wagle, S.R. and Ingebretsen, W.R., Jr. (1975) Methods in Enzymology, Vol. 35, 579-595, J.R. Lowenstein, Ed. Wagle, S.R. and Ingebretsen, W.R., Jr. (1975) Proc. Sot. Expt. Biol. and Med. 149, 480-485. Hugget, A. and Nixon, S.N. (1957) Lancet 2, 268-274. Monier, D., Santhaman, S. and Wagle, S.R. (1972) Biochem. Biophys. Res. Comm. 46, 1881-1886. Berry, M.N. and Friend, D.S. (1969) J. Cell. Biol. 43, 506-520. Capuzzi, D.M., Rothman, V. and Margolis S. (1974) J. Biol. Chem. 249, 1286-1294. Schreiber, M., Schreiber, G., and Kartenbeck, J. (1974) Can. Res. 34, 2143-2150. Wagle, S.R. and Ingebretsen, W.R., Jr. (1973) Biochem. Biophys. Res. Comm. 52, 125-129. Akpan, J.O., Gardner, R. and Wagle, S.R. (1974) Biochem. Biophys. Res. Comm. 6l, 222-229. Wagle, S.R. and Sampson, S. (1975) Biochem. Biophys. Res. Comm. 64, 72-80. Wagle, S.R., Ingebretsen, W.R., and Sampson, L. (1973) Biochem. Biophys. Res. Comm. 53, 937-943. Zahlten, R.N.,Stratman, F.W. and Lardy, H.A. (1973) Proc. Natl. Acad. Sci. 70, 3213-3217. Christoffersen, T. and Bert, T. (1974) Biochem. Biophys. - Acta 338, 408-417. Tolbert, M.E.M., Butcher, F.R. and Fain. J.N. (1973) J. Biol. Chem. 248, 5686-5697. Garrison, J.C. and Haynes, R.C., Jr. (1973) J. Biol. Chem. 248, 5333-5342.

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