J. Nutr.

Antiobesity ridase

and

Antidiabetic

Inhibitor

Hiroyuki

Actions

in Genetically

ODAKA, Akio and Takao

Sci. Vitaminol.,

of a New

Obese-Diabetic

5HIN0, Hitoshi MATSUo

Potent

38, 27-37,

1992

Disaccha-

Mice,

KKAy

IKEDA,

Biology Research Laboratories, Takeda Chemical Industries, Ltd., Jusohonmachi, Yodogawa-ku, Osaka 532, Japan (Received July 15, 1991)

Summary AO-128 is a potent and structurally novel inhibitor of the intestinal disaccharidases, such as maltase and sucrase. Genetically obese-diabetic mice, KKAy, were used to examine the acute or long-term effectiveness of this compound. AO-128 decreased a postprandial rise in blood glucose after sucrose solution loading dose-dependently; the ED50to reduce a delta increment of blood glucose by 50% was 0.22mg/kg. The intestinal sucrase and maltase activitiesswere suppressed to 7 and 48% of the control levels, respectively, at a dose of 0.21mg/kg. Four-week-old female KKAy mice were kept on a laboratory diet containing 10 or 50 ppm of AO-128 for 12 weeks. The high dose of AO-128 reduced food intake and body weight gain throughout the experimental period. On the other hand, the low dose reduced body weight gain for the first 4 weeks without any effect on food intake. Development of the hyperglycemia and hyperinsulinemia characteristic of KKAy mice was moderately prevented by the low dose, and completely by the high dose. Hypertriglyceridemia tended to be suppressed by the AO-128 treatment. The high dose decreased the hemoglobin A 1 level and parametrial adipose tissue weight. Hepatomegaly and fatty liver were ameliorated by AO-128 dosedependently. Nephropathy was ameliorated by the high dose. These findings indicate that AO-128 may be useful for treating human obesity and diabetes. Key Words disaccharidase inhibitor, obesity, diabetes, KKAy mice, hy perglycemia, hyperinsulinemia, sucrase, maltase

A steep rising in the postprandial blood glucose level is a therapeutic problem in carbohydrate-related disorders, such as obesity, diabetes mellitus, and hypertriglyceridemia (1, 2), because the high blood glucose level accelerates lipogenesis and fat accumulation through hypersecretion of insulin, and increases insulin require ment. Recent research indicates that alpha-glucosidase inhibitors, which delay 27

28

H.

Fig,

1.

ODAKA

Chemical

et al.

formula

of AO-128.

absorption of carbohydrates by inhibiting conversion of carbohydrates to monosaccharides, are useful to treat the above-mentioned metabolic diseases (3-7) . On the basis of its inhibitory actions on the porcine alpha-glucosidase activ ities, 1 L-[1(OH), 2,4,5/3]-5-[2-hydroxy-l-(hydroxymethyl)-ethyl] amino-l-C(hydroxymethyl)-1,2,3,4-cyclohexanetetrol (AO-128, Fig. 1) has been selected as one of the promising candidates from a series of valiolamine derivatives (8) . This compound strongly suppresses maltase and sucrase activities of the rat intestinal sucrase-isomaltase complex, but does not suppress the alpha-amylase activity of porcine pancreas (9). The present article describes the antiobesity and antidiabetic actions of this compound in genetically obese-diabetic mice , KKAy (10). MATERIALS Animal. oratory were

The Unit

maintained

on

23.6%

protein,

Japan

Inc.,

the

long-term every

Food

efficiency every

4

(20ƒÊl)

were

taken

in

powdered

a CE-2

a powdered

a

light

diet

the

being an

glucose after the

Antiobesity

before

glucose, sucrase

sinus

increments

antidiabetic

(g)

before

was

the

mice

, Clea

intake

, the start

the

weighed

sifting

per

Lab

(CE-2 food

3 days

and

the

carbohydrate,

off

food

in the

mice

the

feces

intake

.

(g)•~

were

temperature

for

oral

sucrose

18h,

housed

(23•}1•Ž)

,

At

0,

30,

60, basal

activities actions.

load, the

and

end in

groups

of

and

120min of

the

where

the

small

Four-week-old

intestine

or

after

samples loading

plasma

the

the

as

to

glucose

at

were described

female

J. Nutr.

were the

levels

mice

10con-

a represents

c are

test,

9-

Blood

levels

b and

of

(2 .5g/l0ml/kg)

respectively).

from

glucose

. 3

load

(mg/dl•Eh), oral

maltase

vitamins

group

controlled

1.89mg/10ml/kg,

orbital

respectively. and

and

an

and

24h

gain

fasted

as: ƒ¢area=(a+2b+c)•~1/4-a

plasma

These

52 .7%

experiment,

with

in

measure

each

weight

bred age.

of

diet

in

of

(08:00-20:00h).

and

Delta

to

for

the

received

0.63,

order

temperature

room

After

mice

from

to

body

were

weeks

minerals In

as

4

consisting

6.6%

Throughout

test.

glucose.

and

calculated

and

KKAy

diet

at

libitum.

at room

cages

(0.21,

blood

and

weeks.

tolerance

AO-128

examine

changed

was

metal

female

calculated

fiber, ad

KKAy,

weaned

chow

4.9%

drying

(55•}5%),

taining

60min

or in

Sucrose

of

after (%)

2

week-old

measure

was

mice,

and

water

experiment,

week

individually humidity

fat,

diet

Division

laboratory

and

METHODS

obese-diabetic

our

a

4.4%

chow

lump

100

of

Tokyo)

laboratory of

genetically

Animal

AND

KKAy

Sci.

level

30

and

killed below mice

Vitaminol.

to . ,

DISACCHARIDASE

weighing or

13.4-21.7g,

50

(high

were

taken

liver

the

in

ice-cold The

of

ƒÊ

part

of

mixture Lowry

et al.

lipid

with

The above

was

incubated

(12).

maltase

was

fixed

in

intestine

cecum

10ml

was

of

1h.

from

the

tissue as

neutral

we

formalin,

examine

the

or

or

content

maltase

part

progress

just

maltose

in

determined

were and

with

homogenizer. 40mM

was

activities maltose,

below

rinsed

a polytron

sucrose

protein

sucrose

the

longitudinally,

using

40mM

The and

between cut

saline

with

for

in

adipose

examined

10% to

glucose,

activities The

composition

dose)

(100ƒÊl)

plasma

experiment.

was

small

Sucrase

converted

measure

and

(low

samples

(10).

the

with

37•Ž

to

hematoxylin/eosin mice

just

at

the

10

Blood

sucrase

of

29

containing

weeks.

4 weeks

kidney

KKAy

homogenized

concentration

by

calculated

by

indicated

as

were

protein/h.

Analytical

procedures.

reaction

termined

mixture,

The and

enzymatically

Plasma

insulin

Randle

(13)

a standard.

measured

EncoreTM by

Al

Co.). assessed

glucose

The using

concentration

triglyceride

a commercial

Hemoglobin

was

the

using

was using

Nihon-Chemical means

in

DIABETES

diet

12

and

end

liver

activities.

the

and

Al,

right

stained

maltase

reaction

mol/mg

the

and

for 2 or

the

the

The

(5-20ƒÊl)

method

glucose

and

AND

powdered

every

at

(11).

and

saline,

sinus

present

homogenate

0.5ml the

weighed

and

a CE-2 AO-128

Hemoglobin

paraffin,

duodenum

on of

determined

nephropathy

Sucrase the

insulin.

previously

embedded

kept (w/w)

orbital

were were

described

of

the

and

intestine

and

ppm

from

triglyceride, small

were

dose)

INHIBITOR

the kit

was

(Baker double-antibody

significance range

method

differences

plasma

plasma

and

were

Allentown,

based with

a commercial of

blood, the

Co.,

England) using

multiple

the in

Instruments

(Amersham,

determined

statistical

Duncan's

in

concentration

on

human kit

de PA).

Hales

and

insulin

as

(NC-ROPET, between

sample

test.

RESULTS

Effect on postprandial hyperglycemia AO-128 dose-dependently decreased the postprandial rise in blood glucose after an oral sucrose load (Fig. 2). The ED50 value required to reduce a delta increment of the blood glucose area by 50% was 0.22mg/kg. The intestinal sucrase and maltase activities measured 120min after AO-128 load were suppressed to 7 and 48% of the control levels even at the lowest dose; further suppression of these activities by increased dosages of AO-128 was small (Table 1). Antiobesity and antidiabetic actions Four-week-old female KKAY mice were kept on a CE-2 powdered diet con taining 10 (low dose) or 50 (high dose) ppm (w/w) of AO-128 for 12 weeks. The daily intake of AO-128 calculated from food intake every week changed from 2.5 to 1.4mg/kg/day in the low dose group and from 9.6 to 7.4mg/kg/day in the high dose group with advancing age. The high dose of AO-128 induced diarrhea and soft feces only within the first 5 days; the low dose did not. The high dose reduced food Vol.

38, No.

1, 1992

30

H. ODAKA

Fig.

2.

Effect

for

18h,

of AO-128 9-

sucrose

or

or

1.89

range

Table.

1.

maltase

activities

Effect

being

fasted

small

was

intestine

values

body of

gain

and

weight food

intake.

demia,

and

of

globin in

Al

the

high

significantly 3.1•}0.4g, from

the

at

lighter

in

p