J. Nutr, Sci. Vitaminol.,

Diurnal

Variation Activity

and

Increase

in Diabetic

38, 265-276,

1992

of Disaccharidase Rats

Kazutaka NASHIRO,Keiji MURAKAMI,and Goro MIMURA Second Department of Internal Medicine, School of Medicine, University of the Ryukyus, Okinawa 903-01, Japan (Received December 27, 1991)

Summary The small intestinal disaccharidase activity and its daily variation in the diabetic rat have not been well described. Therefore, the small intestinal disaccharidase (maltase, lactase and sucrase) activity and its daily profile were studied in streptozotocin-induced diabetic rats under physiological conditions. In diabetic rats, a similar pattern of diurnal variation of disaccharidase activity to control rats was observed, while the relationships between daily change of disaccharidase activity and that of food consumption suggested that there was a different mechanism of diurnal variation in diabetic rats. On the other hand, a significant increase of mean 24-h lactase and sucrase activities was noted in diabetic rats, while that of maltase was not significant. Using the in vitro incubation method, a significant correlation between glucose concentration and lactase or sucrase activity but not maltase activity was observed. How ever, insulin showed no effect on disaccharidase activity. Thus we clarified the presence of a diurnal variation of disaccharidase activity and an increase in its activity in diabetic rats. This change was suggested to be derived from high plasma glucose level. Key Words disaccharidase activity of the small intestine, maltase, lac tase, sucrase, diabetes mellitus, diurnal variation, food consumption, plasma glucose, insulin, rats

It has been known that disaccharidase (DS) activity in the small intestine in normal rats shows daily rhythmic change related to the time of food ingestion (1, 2). On the other hand, small intestinal DS activity in diabetes mellitus has not been well understood. Some authors have reported increased DS activity in diabetic rats (3-11), but conflicting observations also have been published (12, 13). In diabetic patients no consistent opinion has been presented concerning the small intestinal DS activity (14-17). In the present study, we examined the DS activity and its daily profile in the 265

266

K. NASHIRO

et al.

small intestine of streptozotocin (STZ)-induced diabetic rats. For the purpose of clarifying the mechanism of daily variation of DS activity, its relationship with the amount of food ingestion and plasma glucose level was examined. Furthermore, to study the mechanism of increased DS activity, direct effects of glucose or insulin on DS activity in the small intestinal mucosal cells of rats were also examined in vitro. MATERIALS AND METHODS Male from

Wistar

rats

to

19:00h,

7:00h

and

a

room

given

ad

One

group

glucose

levels

plasma

glucose

At the

every

4h

48

the

from

(HbA1)

determination. was 1.

4

excised membrane with

in

Enzyme

water

were

the

cell

York, 10min, than

and

Chemical

the

were

enzyme of

The water

both

and

was

Ltd.,

the

solutions reaction for

the

measurement).

prepared

enzyme bath

Then

2 min.

Osaka, at

substrate were was

mixed stopped

Liberated

by

solutions

and

by glucose

4

the

rats

blood

was

hemoglobin insulin

on

DS

rats.

every

-40•Ž Dahlqvist

parts

of

buffer

were

incubated

for

mixture

was

immersing

the the

by

scraping

al.

chilled

(19)

distilled

Inc.,

(0.1M, 5min

at

Ltd., pH

for

less Pure

Kyoto,

6.0).

Both

Then

15min

at

mixture

in

determined J. Nutr.

for

to

(Wako

at 37•Ž.

was

New 4•Ž

(diluted

lactose Chemicals

reaction

analysis. et

3,000•~g

incubated

substrates

the

mucous

[MicrosonTM

solution

(Nakarai

under

the

until

of

[maltose,

maleate

At

4h washing

5min

at

enzyme

sucrose

from

group.

time,

ultrasonication

sodium

the

had

control

collected

at

centrifuged

as

solutions

each

Systems-Ultrasonics,

collected

in

in

After

was

method

homogenized

Japan),

we

control

within

Namely,

was

and excised

stored

modified

mixture

showing

42

or

rats.

intestine

(Heat

rats

glycated

diabetic

control

were

MS-50

same

glucose

were

Substrate

0.056M

of

saline,

small

the

was

Model

90s.

in

42

membrane.

tissue

supernatant at

industries

Japan)]

the

the

by

mucous

and

for

0.10U/ml

determined

frozen

profile

specimens

U. S. Plasma

vitro.

physiological of

other

Louis,

Thus

and

and

effect

in

and

those

4 rats

the

glucose

intestine

diabetic

chilled

collected

disruptor,

U. S. A.)]

its daily small

42

with diabetic

At

direct

the

the

St.

diabetic.

42

plasma

and

as

studied

from

rats

and

two

weeks.

activity.

the

were

into

injection. and

defined

water

divided

Co.,

(light

humidity

and

intraperitoneal

was

6 control

50•}5%

group)

injection,

12

cycle

chow

Chemical

by

of

for

with

(control

were

DS

light-dark

randomly

(Sigma

collected

vein

one-third the

was

added,

ultrasonic

the

upper and

activity thawing

ƒÊl

the

glass,

after

in with

buffer

ingestion

of

caval

treatment,

intestine

a slide

age

profile

using

anesthesia small

the

was

activity

the

pentobarbital

food

were

after

more,

12-h

7:00h),

rats

week or

a

laboratory

group)

Furthermore,

investigated

after

of

vena

Disaccharidase

weeks

at

daily

inferior

activity

rats

mucosa

the

weeks, citrate

one

profile

to

80mg/kg

250mg/dl

control

study

19:00h

received

determined

intestinal

to

8

(diabetic

daily

small

collected

of

(18)

of

of

Standard

buffer

levels

and

from

age

rats

citrate

were

first

Then

of

conditions

24•}1•Ž.

streptozotocin in

diabetic

the

under

dark

of

group

dissolved

42

and

At

received

A.)

kept

temperature libitum.

groups.

were

Sci.

100 37•Ž.

a boiling by

the

Vitaminol.

DIURNAL

glucose the g

VARIATION

oxidase

enzyme of

method,

protein

was by

Nakarai

of

DS 2.

each

was

Daily

were

time 00,

12:00,

per

gram

rats

in

was

profile

mucosa

was

collected,

Plasma

glucose Test, affinity

Tokyo,

daily

level

vena

Co.,

cava the

in

in

inferior

22)

ingestion

in of

starting

food

from

04:00,

08:

expressed

groups.

same

control time

rats.

when

using

and

rats 3days

times

syringe.

method

(GLU

hemoglobin

GHb,

of

intestinal

a heparinized

glycated

(Glyc-Affin•

Blood

the

oxidase-mutarotase

Japan),

(21,

4h

of both

vein

glucose

for

at

and

the

Four

kept

every

pattern

estimated.

food

diabetic

at

daily

also

of

pattern

4h,

Tokyo,

method

consumed

was

Fraction

rats.

were

of per

protein

the

was

control

they

amount

compared

every

by

Test

column

Effect

control

rats.

control

rats

of glucose The

in

3.3mM

glucose. The

15min

at

37•Ž.

containing was

pre-incubated

and

its

(3.3,

8.3,

was in

16.7

Denmark;

0,

pre-incubation After

10,

100,

in

(HbA1)

Seikagaku

were

variation

of

The bation

cell

DS

periods 5.

Kogyo,

carried

out

activity

was

was of

analysis.

daily

profile

calculated

p

values

Vol.

3, 1992

38, No.

found

the

are

small using

of two-tailed.

0.5%

BSA

to

be

The

Student's was All

t-test

values

cells

test

compared p

02

glucose various

Bagsvaed, above, for

after 20min.

above. when

for

95%

with

glucose

sus

These

the

diurnal

small.

mucosal

dye-exclusion

values

of

A/S, as

with

cell

of

described

20:00h,

3.3

glucose

phase

manner

16.7mM

and

relatively

intestinal

3.3mM gas

6

for

times

this

incubated

method

17:00h

4 of

Nordisk

same and

BSA,

Germany)

concentrations also

of

containing

0.5%

and

Novo

the

(KHBB)

the

from

immediately

washed

BSA

was

the

was

aliquot

under

cells intestine

GmbH,

various

by

between

examined

The

0.5%

7.4

was

Insulin,

measured

It

An

3h

in

small

containing

precipitate

suspension

1,000ƒÊU/ml)

mucosal

the

Manheim,

for

Human

was

pH

KHBB

containing cell

of

above.

glucose.

37•Ž

containing

Statistical

values.

The

and

of

containing at

KHBB

activity

viability

10ml

intestinal

1/3

buffer,

the

KHBB

the

described

3.3mM

(Actrapid

KHBB DS

in

and

33.3mM). insulin

of upper

(Boehringer

incubated BSA•

of

incubation,

incubations

in

0.5%

and

treated

BSA

then

the

method

centrifugation,

0.5%

concentrations

the

activity

of

collagenase

After

20min,

DS

bicarbonate

was

1mg/ml

pension

CO2

by

tissue

KHBB

on

membrane

Krebs-Henseleit

minced and

insulin

obtained

chilled

glucose

5%

or

mucous

was

minced

and

glucose

the

food

of albumin,

Thus

and

activity activity

amount

activity

and

the

267

Japan). 4.

mM

The

determined

Shino

of

was

collected from

diabetic cages,

RATS

enzyme

serum

standard. DS

as

the

The

the

ingested

defined

and

(bovine

24-h

in

the

hour

of plasma

was

as

IN DIABETIC

was

activity.

mean

24:00h.

was

a minute,

BSA

amount be

per

activity

in

Japan)

individual

to

DS

using

the

mean

weight

group

Neo-Shino

the

ACTIVITY

specific

ingestion

in

20:00,

body

Daily

the

and

considered

each

by

Kyoto,

of food

16:00, of

3.

Ltd.,

Then,

0:00h

of

(20)

examined

isolated

acclimatization.

the

method

profile

group

unit substrate

as

Lowry

Chemicals

activity

one

1ƒÊmol

expressed

the

DISACCHARIDASE

and

hydrolyzing

measured V,

OF

by was by

less

than

throughout

trypan used

the

incu

blue. to

compare

analysis

of

0.05

were

variance. considered

mean All to

268

K. NASHIRO

indicate

statistical

significance.

All

et al.

group

data

are

reported

as

mean•}SE.

RESULTS In 3.4g

the

diabetic

4 weeks

increased in

after

from

the

was

Disaccharidase

in

group)

protein

in

both

and

from in

the

the

in

208.8•}1.0g

control

same

(p