Vol. 183, No. 2, 1992

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

March 16, 1992

Pages 814-820

INSULIN

STIMULATES RAT

NOVEL PROTEIN ADIPOCYTES

KINASE

C IN

Tatsuo Ishizuka,’ Mayumi Yamamoto, Kazuo Kajita, Toshihiko Nagashima, Keigo Yasuda, Kiyoshi Miura, Denise R. Cooper*, and Robert V. Farese* The Third *James

Received

Department Medicine,

A. Haley

February

of Internal Medicine, Gifu University Tsukasa-machi 40, Gifu 500, Japan

Veterans

3,

Hospital, 13000 Bruce Tampa, Florida 33612

B. Downs

School

of

Boulevard,

1992

Summary: Insulin is known to rapidly stimulate and/or translocate Ca2+/phospholipid-dependent protein kinase (conventional PKC; cPKC) in rat adipocytes. Presently we examined whether insulin also stimulates/translocates Ca 2+-independent, phospholipid-dependent protein kinase (novel PKC; nPKC). Total Mono Q column-elutable nPKC (like cPKC) activities were decreased in cytosolic and increased in membrane fractions with insulin treatment. lmmunoblot study of novel PKC E also showed insulin-induced from cytosol to membrane, translocation of immunoreactive PKC similar to the translocation of cPKC, PKC 6 . These results suggest that nPKC has an important role in insulin-induced signal 0 1992AcademicPress, Inc. transduction.

The role of phospholipid-dependent signal transduction mechanisms during insulin action has recently been studied (i-4). Insulin increases diacylglycerol(DAG) production by stimulating the synthesis of phosphatidic acid (PA) de nova (1, 5-7), and the hydrolysis of phosphatidylcholine(8) and a phosphatidylinositol glycan (9). Whatever the source of DAG, activation and translocation (PKC) from cytosol of Ca2+/phospholipid-dependent protein kinase

1

To whom correspondence

should

be addressed.

12-0-tetradecanoyl phorbol-13-actate, Abbreviations used : TPA, EGTA, ethylene glycol bis (O-aminoethyl ether)-N,N,N’,N’-tetraacetic acid, PMSF, phenylmethylsulfonyl fluoride. 0006-291X/92 $1.50 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

814

Vol.

183,

No.

BIOCHEMICAL

2, 1992

to membrane

appear

subsequently

result

AND

to occur in

BIOPHYSICAL

rapidly

increases

RESEARCH

in rat

in

50

COMMUNICATIONS

adipocytes

KDa

(3,

catalytic

4) and

fragment

of

PKC during insulin treatment in rat adipocytes(l0). Molecular cloning experiments as well as biochemical characterization of PKC, have established that Ca2+/phospholipid-dependent, conventional PKC (cPKC) constitutes a protein related proteins, including PKC

family of at least four closely a , 81, 811 and y, which are

expressed

in a cell

type-specific

manner

(ii-1

additional

members

of the

family,

i. e.,

including

PKC

E, 6, and 6

Ca2+-independent, conventional the

PKCs,

identification

except enzyme

for

conventional

nPKC activity PKC

PKC (16-18)

which

also

been

The

proteins

has

not

been

E (16,17).

In this

study

we

have

during

PKC insulin

Materials

E

distinct

at the cDNA

of these and

immunoreactivity, treatment

PKC

of

(nPKC),

up a subfamily

PKCs

found

presence

novel

make

phospholipid-dependent has

5).

from

level, yet

of the

although

established

examined as

nPKC well as

in rat adipocytes.

and Methods

Materials Pork insulin was obtained from Novo (Copenhagen, Denmark). [y32P]ATP (3000 Ci/mmol) was purchased from New England Nuclear (Boston, MA). Phosphatidylserine (PS), diolein, histone (type Ill-S), phenylmethylsulfonyl fluoride (PMSF), leupeptin, 12-Otetradecanoyl phorbol-13-acetate (TPA), BSA, 2-deoxyglucose (2DOG), D-glucose and ATP were obtained from Sigma Chemical Co. (St.Louis, MO). Collagenase (Type I) were obtained from Worthington Biochemical(Freehold, NJ). All chemicals were reagent grade or better. Tissue preparations for in vitro studies Rat adipocytes were prepared as previously described (2, 3). These suspensions were allowed to equilibrate at 37°C for 30 min before incubation with agonist. In PKC studies insulin (10 nM) or vehicle (controls) were added in a retrograde sequence over the course of a 20 min period which was constant for all samples. Reactions were terminated by addition of 10 ml ice cold buffer I (20 mM Tris-HCI, pH 7.5 containing 0.25 M sucrose, 1.2 mM EGTA, 0.1 mM phenylsulfonyl fluoride, 20 pg/ml leupeptin, and 20 mM 2mercaptoethanol). The adipocytes were washed twice , homogenized in buffer I and centrifuged to obtain cytosol and membrane fractions. Measurement of [3H]2-DOG uptake in vitro We routinely observed 6-8 fold increases in [3H]2-DOG uptake following insulin treatment as previously described (2)(Data not shown). Protein kinase C (PKC) assay PKC was analyzed by histone phosphorylation assay of Mono Q column-purified PKC and immunoblotting, as described previously (2, 815

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3, 19). Column fractions were analyzed histone phosphorylation activity of Ca 2+(0.5 mM)/PS (40 pg/ml)/diolein (0.4 pg/ml)/, Ca2 + alone, EGTA(0.5 mM)/PS/diolein and EGTA alone as modified previous procedure (2, 3, 19). All reaction mixtures (250 ~1, total volume) contained 20 mM Tris-HCI, pH 7.5, 5 mM magnesium acetate, 0.02 % histone, 10 uM [y- 32P]ATP ( 25-30 x 104 cpm/nmol), and 100 pl column fraction. After incubation for 5 min at 3O”C, the reaction was stopped with 25 % trichloroacetic acid. Acidprecipitable materials were collected on membrane filters and counted for radioactivity. Basal activity, measured in the presence of 0.5 mM EGTA, was subtracted from Ca2+/PS/diolein/-dependent protein kinase activity to determine cPKC activity. Basal activity was also subtracted from EGTA/PS/diolein-dependent protein kinase activity to determine nPKC (20). PKC was also analyzed by immunobloting as previously described (2, 3, 19). Results

Elution MonoQ

profile column

of conventional

PS/diolein/Ca2+-dependent eluted

as

shown

cytosol

from

peaks

(This

previous

in Fig 1, during there

elution

report

4

protein

rat adipocytes:

independent,

2

PKC

(3),

profile due

is

6

10

kinase

slightly

to a different

12

novel

PKC

activity

(cPKC)

activity

using was

MonoQ column chromatography of were two larger, and one smaller

phospholipid-dependent

6

and

different elution

protein

14

from

that

procedure). kinase

2

6

6

10

Fraction

Fraction

Fig. 1. Elution profile of PKC using MonoQ column. Cytosolic protein (10 mg) from epididymal fat pads applied onto MonoQ column as described in “Materials and Methods”. (A) Conventional PKC activity ( q ) was measured by subtraction of EGTA-kinase activity from Ca2+/PS/diolein kinase activity. (B) Novel PKC activity (a) was activity from measured by subtraction of EGTA-kinase EGTA/PS/diolein-kinase activity. Similar results were obtained in three separate experiments. 816

a

Ca2+activity

(nPKC)

4

of

12

14

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BIOCHEMICAL

2, 1992

AND

BIOPHYSICAL

80 KDa

RESEARCH

anti-PKCB

antibody

anti-PKC 3

5

4

6

7

8

9

COMMUNICATIONS

epsilon

antibody

Fraction

10 11

Fig. 2. lmmunoblot analysis of cPKC 0 and PKC E. After passing through MonoQ column as described in “Materials and Methods”, each Centricon Tube. Equal eluted fraction was concentrated by using fraction (4 ul) was applied to SDS-PAGE, amount of cytosolic transferred to nitrocellulose membrane, incubated with anti-PKC 0 or -PKC e (GIBCO, NY) as described in “Materials and Methods”. Similar results were obtained in three separate experiments.

eluted

as

two

different

from

lmmunoreactivity We

also

of cPKC

of cPKC

by

greatest

intensity

translocation

12),

decreased

PKC

of

by

by

100 % after

described calculated

slightly

fractions using

fraction

anti-PKC

(fraction

column

6 were

3 to

(Fig.

observed

2).

in fractions

5

decreased

by

%

40

calculated

of insulin

the

treatment

hand,

in

all

total

of cytosol

and

E antisera. Cytosolic membrane-associated 817

sum

fractions

activity (Fig.

3-a))

elutable

as

nPKC

(

3 to

12)

nPKC activity (Fig. 3-b)).

E in rat

adipocytes

membrane PKC

of

phospholipid-

membrane-associated 10 min of insulin treatment PKC

and

in all fractions

membrane-associated

other

I3 and

cPKC

the

activity

of Ca2+-independent,

activity

of PKC

of

as

kinase

10 min On

treatment of insulin Fig. 4 shows an immunoblot 13 and -PKC whereas

activity

and

by 51 % after analysis

enzymatic

and

sum

4

6.

50 %,

(3). as the kinase

anti-PKC decreased,

nPKC

MonoQ

protein

protein

lmmunoblot

were

Q column

and

in each

activity

dependent increased

Mono

in fraction

Ca2+/phospholipid-dependent

activity,

in

through

of 80 KDa

in rat adipocytes elutable cPKC

previously

which

the greatest activity in fraction 5. In comparison, bands of 90 KDa PKC e were observed in fractions

Insulin-induced

increased

peaks,

of cPKC

respectively

passing

bands

with

( 3 to

smaller activities.

and nPKC

E antibodies,

to 10, with immunoreactive

one

immunoreactivity

followed 111, lmmunoreactive

nPKC Total

and

peaks

showed

0 and -PKC

to 7,

larger,

fractions

during using

p immunoreactivity immunoreactivity

Vol.

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2, 1992

BIOCHEMICAL

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BIOPHYSICAL

RESEARCH

COMMUNICATIONS

300, l

b

a

100n Membrane

Cytosol

Membrane

Fig. 3. Insulin-induced translocation of cPKC and nPKC activities in rat adipocytes. Total elutable PKC activity was determined after purification of the enzyme by chromatography on the MonoQ column ( see Fig. 1). Cytosolic (open bar) and membrane-associated (closed bar) fractions of control value were indicated as 100 %. Insulininduced percent change was expressed as mean f SE of 2-3 results (* ; p < 0.05 by paired ttest). Panel a) indicated mean of % change of conventional PKC activity (3 results), and panel b) indicated mean of % change of novel PKC activity (2 results) in control and insulinstimulated value.

increased KDa

PKC

membrane

during

treatment

E also

with

decreased

in the

cytosol

and

increased

R immunoreactivity immunoreactivity,

even

to

Membrane-associated

though

larger

amounts

of protein

immunoreactivity

I3 and PKC E was maximal at 10 min after treatment

in the

anti-PKC

anti-PKC

5

10

were

used

of both PKC

of insulin.

Membrane

CytOSOl

0

90

Note that the intensity of 80 KDa cytosolic PKC PKC E higher than 90 KDa of cytosolic was

fraction.

assay PKC E.

lmmunoreactive

10 nM insulin.

20

0

5

IO

20

B antibody

E antibody

min of treatment

with

insulin

Fig. 4. lmmunoblot analysis of PKC 0 and PKC E in rat adipocytes during treatment of insulin. Upper panel indicates changes in 80 KDa PKC I3 immunoreactivity and lower panel indicates changes in 90 KDa PKC E immunoreactivity of each cytosol and membrane fraction at the indicated times (0, 5, 10 and 20 min) during treatment of IO nM insulin as described in “Materials and Methods“. Similar results were observed in at least three other experiments. 818

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Discussion Recent studies,

molecular as well

have

revealed

related

cDNA E and

clones the

from

other

subtype has been In this study, cytosolic

a rat brain

two

being

immunoreactivity. PKC

were

of both cPKC

measured

nPKC

kinase

activity,

measured

kinase

activity

measured

diolein.

Ohno

et al. (20)

is fully

activated

5 does

have

failed

by

and

nPKC

enzyme

activity

in the

not require to

have

phorbol

find

recently,

that

being the q-

decreases in activity and PKC E

to insulin-induced

changes

insulin

in

stimulates

the

in rat adipocytes. using

presence

presence

the subtraction of 0.5

mM

of 0.5 mM

reported ester and

that purified PS without

Ca 2+ for activation

rapid

one of them

enzyme

suggesting

in the

library,

provoked

nPKC

similar

activation,

We

PKC

by cDNA expression at the protein level,

6 and 5. More

insulin

in membrane

These

conventional

cDNA

PKC

defined (18). we found that

and increases

translocation

followed of PKC

the presence of at least four distinct conventional CI, 01, 011, and y, which were closely related In addition, Ono et al. isolated other novel PKC-

PKC types, sequences(ll-17). PKC

cloning experiments as detailed analysis

insulin-induced

(16).

of basal EGTA,

EGTA, nPKC Ca2+.

Many

increases

from

PS and E activity Similarly

investigators in

intracellular

Ca2+ in insulin sensitive tissues. Thus, in the absence of changes in intracellular Ca2+ concentration, insulin still translocates both cPKC

and

nPKC

in adipocytes.

Recently,

it has

been

nPKC E is stimulated by insulin in cultured fetal chick however, both cytosolic and membrane-associated increased observed

without apparent the translocation

rat adipocytes. induced column

decreases

PKC activity

due to

contamination

translocation of nPKC E. In contrast, we of nPKC E from cytosol to membrane in

versus

observed

with

of cytosols

chromatography

insulin-induced

(22).

increases

DEAE-chromatography,

with DAG or other Therefore,

insulin-induced translocation of nPKC be observed by assaying PKC activity column

neurons (21): nPKC activity

reported that differences between insulinof cytosolic PKC activity observed with Mono Q

chromatography,

DEAE

that

We have

cytosolic after

reported

of

may be

PKC activators

it is possible

that

E in fetal chick neurons may after purification by Mono Q

chromatography

In summary, we suggest that in addition to conventional Ca2+/phospholipid-dependent protein kinase, novel PKC also plays a role in insulin-stimulated signal transduction mechanism in insulinsensitive tissues. 819

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References 1.

2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

12. 13.

14. 15. 16. 17. 18. 19. 20. 21.

22.

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