Inhibition of glucose absorption by phlorizin affects intestinal functions in rats

GASTROENTEROLOGY

1993;105:692-697

Inhibition of Glucose Absorption by Phlorizin Affects Intestinal Functions in Rats HISANORI YUKIHIRO

MINAMI, JI-RYUN KIM, KAYOKO NAKABOU, KENTARO SAKAI, and

TADA, FUMIE TAKAHASHI, HIROSHI HAGIHIRAT

KEN-ICHI

MIYAMOTO,

Department of Nutrition, School of Medicine, The University of Tokushima, Tokushima, Japan

Background: To investigate the mechanism of regulation of intestinal disaccharidase activity and glucose absorption, the effect of dletary intake of phlorizln, a potent and specific inhibitor of intestinal glucose transport, on intestinal disaccharidase activity and Na+-dependent glucose transporter was examined in rats. Methods: Jejunal disaccharidase activity and the number of Na+-dependent glucose transporters were determined in rats maintained on a low-starch diet, a high-starch diet, or low-starch diets containing various amounts of phlorizln (O.l%-0.9% wt/wt). Results: Jejunal dlsaccharidase activity increased in a dose- and time-dependent manner. Stimulation of jejunal disaccharidase activity only occurred when phlorizin was added to starch-containing diets, not when it was added to a carbohydrate-free diet. Additlon of the same amount of phloretin and glucose (constituents of phlorizin), to the diet failed to increase disaccharidase activity. The maximum binding of phlorizln to brush border membrane vesicles was increased in the rats fed phlorizin, whereas the dissociation constant remained unchanged, suggesting an increase of glucose transporter expression. Conclusions: Dietaty phlorizin increased the jejunal dlsaccharidase activlty and Na+dependent glucose transporter expression. The trigger for these changes may have been due to an increased luminal glucose content.

and the number brush

dependent charidase

1

of a high

carbohydrate

diet is known

to

cause a parallel increase in intestinal disaccharidase activityye6 and sugar absorption.7-9 Induction of disaccharidases is not only produced by a diet with a high content of disaccharides or starch but also by a diet containing monosaccharides such as glucose, galactose, fructose, or 3-o-methyl-glucose.3,5,6 The finding that the specific activity of sucrase increases in proportion to the dietary starch content”,” suggests that the sugar leve1 in the intestinal lumen influences disaccharidase activity. Glucose is absorbed via a specific transporter that is located on the brush border membrane of the intestinal enterocytes.12 Because carbohydrate loading causes a paralleled increase in disaccharidase activity

of glucose

membrane,

inhibitor

used to measure Because

in the intestinal of both

of the glucose

glucose

transporter

of its marked

porter,

carriers

the induction

Na+-

glucose transporter expression and disacactivity is likely to be closely related. Phlori-

zin is a specific

inhibition

we investigated

on intestinal to investigate

function. whether

affected

intestinal

pendent

glucose

carrier13 and is

density

in vitro.14

of the glucose

the effect of phlorizin

trans-

ingestion

The present study was designed addition of phlorizin to the diet

disaccharidase transporter

activity

expression

and Na+-dein rats.

Materials and Methods Animals

and Diets

Six groups of male Sprague-Dawley rats (Japan SLC Inc., Hamamatsu, Japan), weighing 200 g were used. To investigate the effect of phlorizin on jejunal disaccharidase activity, rats were maintained for 7 days on a low-starch diet (LS diet [calorie percent]: 20% casein, 10% starch, 10% torn oil, and 60% cotton seed oil shortening), or a high-starch diet (HS diet: 20% casein, 70% starch, 10% torn oil). In some groups of rats, various amounts of phlorizin (0.1% 0.9% wt/wt) were added to the LS diet. On day 7 at 10 AM, the animals were killed by decapitation, and brush border membrane glucose

ngestion

border

vesicles

equivalent

(BBMV)

were prepared.

ingested

on the last day of feeding

4.9 ? 0.3, 4.4 21 0.3, and wt/day

on the LS diet,

groups,

respectively,

31.4 f

1.4 mmol/lOO

0.9% phlorizin

and the amount

was 0.10 + 0.01 mmol/lOO

The amount

diet,

g body

and HS diet

of phlorizin

g body wt/day

of was

ingested

in the rats fed a

0.9% phlorizin diet. The addition of phlorizin to the diet had no effect on the growth, food intake, and intestinal weight of the rats. NO morphological abnormalities of the intestines or symptoms such as diarrhea were observed. To investigate changes in disaccharidase activity after the ingestion of a phlorizin-containing diet, rats were fed an LS TDeceased. Abbrevlations used in this paper: BBMV, brush border membrane vesicles; B,,, , maximum phlorizin bindlng; CHO, carbohydrate; HS, high starch; b, dissociation constant; LS, low starch. 0 1993 by the American Gastroenterological Association 0016~5065/93/$3.00

September

1993

diet containing diet)

2 mmol

phlorizin

per 100 g (LS-phlorizin

for 1, 2, 3, or 6 days after being

standard

phlorizin

effect of phloretin

of phloretin

was also examined.

termined

after 3 days on this new diet. the effect

or with phlorizin

(2 mmol/lOO

To examine activity

trifuged

g of diet).

of Phlorizin

was determined

Tris/HEPES

buffer

Tris/HEPES,

with

150 mmol/L

(pH 7.5) at room

mixture

at 20,000 X g for 30 minutes,

twice with a suspension

BBMV

NaSCN,

was then cen-

the pellet was washed

(300 mmol/L

mannitol

pH 7.5), and enzyme

activity

Preparation Activities

of BBMV and Assay

Jejunal

were prepared

BBMV of Kessler

smal1 intestine saline.

longitudinally,

et al.”

was quickly

to the ileocecal

ice-cold

and

[3H]phlorizin

After

incubation

the reaction saline.

Toyo,

Tokyo, Japan),

with ice-cold

saline.

was counted

using

removed

The upper

from

was

was scraped

of rats. The activity disaccharidases

were used as substrate respectively) a1.16 and

enzymes,

purity,

phosphatase

in al1

isomaltase,

and maltase,

activity

representing

minute

at 37°C.

Protein

hydrolysis

method

of Lowry

et al. using bovine

protein,

was determined serum

BBMV

were

assessed

To investigate along

the villus-crypt

the

Activity

distribution

various

sub-

axis, intestinal

Along the

epithelial

+ SE or SD. Differwere

determined

and P values

a significant

by

< 0.05 were

differente.

and phloretin

were

(St. Louis,

Ltd.

Chemicals

[3H]phlorizin

purchased

Nuclear

Ci/mmol)

Corp.

(Boston,

from

MO)

Inc. (Downsview, (40.40

used were of analytical

the

and To-

Canada),

re-

was purchased MA). Al1 other

grade.

Effect of Phlorizin on Jejunal Disaccharidase Activity Addition pendent

effect

1). Because data The

of phlorizin on jejunal

the

increase

homogenates obtained

time

course

and

using

activity

cells were

diet) began

is shown to increase

to the diet

disaccharidase BBMV

homogenates of phlorizin

in Table

had a dose-deactivity

of disaccharidase

of the changes

ity due to ingestion of sucrase

groups

Results

jejunal

of Sucrase Axis

plot.

as the

strate concentrations.

Distribution Villus-Crypt

me-

and the disso-

a Scatchard

Company

reagents

per

for jejunal disacchaactivities of the enusing

as the mean

(ANOVA),

from New England

by the

albumin

(B,,)

from

retention

BBMV-free

Analysis

experimental

Research

1 U of

of 1 l.trnol of substrate

content

standard.” When the kinetic parameters ridases were determined, the specific in purified

with

by filtering

(Kd) were obtained

Na+-deof KSCN

Nonspecific

binding

times

by subtracting

Phlorizin

spectively.

for sucrase,

three

counter.

Chemical

ronto

and

as units per milligram

Sigma

and the

palatinose,

trans-

on the filter

in the presence

of NaSCN.

phlorizin

as indicating

maltose

(sucrose,

5 mL of

Materials

as judged

same

was determined by the method of Forstner et respectively. Al1 enzyme activities Dahlqvist,”

were expressed

zymes

was opened

with

retained

scintillation

was determined

of variante

accepted

off onto an ice-

was the

of alkaline

of

clean with

analysis

4

tempera-

was immediately

was determined

Data are expressed

the

pmol/L,

at room

by dilution

bound

by the filter

the ligament

half of the intestine

of marker

killed,

and was flushed

cold glass plate with a razor blade. BBMV

various

of phlorizin

between

(final

150 mmol/L

and the filter was washed

and

ences

containing

(0.5 pmol/‘L-10

The radioactivity

Statistical

et

filter (0.45 l,trn pore size, Advantic

binding

constant

was deterwas mixed

or KSCN,

sample

to

of Toggenburger

for 1 minute

a liquid

phlorizin

Binding

of buffer

was stopped

to a nitrocellulose

the amount

and alka-

suspension

NaSCN

The diluted

ferred

of Enzyme

rats were

volume

pCi/mL).

by the Ca’+ precipita-

After

junction

and the mucosa

by enrichment groups

an equal

mannitol,

pendent

by a previ-

segment

activity,

to BBMV

method

of the BBMV

dium. The maximum

method

Treitz

filtration

75 mmol/L

ciation

tion

of [3H]phlorizin

with

segments

of [3H]Phlorizin

from that in the presence

determined.

sucrase

The binding

rapidly

693

as described

from each intestinal

content,

concentration)

ture,

on BBMV,

by incubating

75 mmol/L

for 1 hour. The reaction

10 mmol/L

on Enzyme

intestinal

of Weiser,‘”

by the rapid

ice-cold

and 10 mmol/L

temperature

phlorizin

DISACCHARIDASE

activity.

a1.14 A 5O+tL aliquot

oil, 70% cotton

the direct effect of phlorizin

phlorizin,

mannitol,

10% torn

mined

everted

collected

Measurement BBMV

starch

to phlori-

an LS, or an HS diet without

In Vitro Effect Activity

1 mmol/L

activity

for 7 days on a carbohydrate-free

diet: 20% casein,

seed oil shortening),

from

line phosphatase

was de-

the dietary

of disaccharidase

zin, rats were maintained

enzyme

of varying

activity

INTESTINAL

of the method

were assayed for protein

diet (2 mmol

per 100 g). Enzyme

on the response

the

After 7 days on the LS

and glucose

diet (CHO-free

and glucose,

to an LS-phloretin

phloretin

To investigate

isolated

modification

ously. zOThe fractions

consists

diet, the rats were changed

AND

successively

on the

LS diet for 7 days.

Because

intake

maintained

PHLORIZIN

2. The

significantly

in

only

the

was similar, are

(Table

activity shown

below.

in disaccharidase

activ-

(2 mmol/lOO

g of LS

disaccharidase

activity

on day 1 (P < 0.05)

and

694

GASTROENTEROLOGY Vol. 105, No. 3

MINAMI ET AL.

Table 1. Effect of a Phlorizin-Containing

Diet on the Activity of Jejunal Disaccharidases Sucrase (mU/mg protein)

n Homogenate Low-starch diet 0.1% phlorizin 0.3% phlotizin 0.6% phlorizin 0.9% phlorizin High-starch diet Brush border membrane Low-starch diet 0.1% phlorizin 0.3% phlorizin 0.6% phlorizin 0.9% phlorizin High-starch diet

Isomaltase (mU/mg protein)

15.9 + 1.2=

25.5 30.7 36.5 43.1 7 1.8 160.7 346.0 396.0 482.6 563.5 768.5

3.9 5.5 7.0 7.2 8.9 14.3

1I o.4a ?Z0.2b + o.7c + 0.3’ & 0.3“ * 0.9=

0.12 0.16 0.18? 0.20 0.24 0.40

36.9 63.1 76.6 85.2 104.3 146.2

+ 2.2= ? 3.0b + 8.2b ?Z4.5’ f 9.3d f 0.9=

0.91

f 0.1 la

1.50 1.65 1.86 2.05 3.40

tr f f + +

f 1.6b ?Z 1.2c ? 2.9’ f 2.1e f 6.8‘ f f f f f rt

10.2= 22.8b 31.1b 32.5’ 63.3’ 92.4e

Maltase (U/mg protein)

NOTE. Rats were fed one of six diets for 7 days. Phlorizin was added to the low-starch diet in some groups of rats (O. l%-0.9%, expressed as the mean f SE. Values in the same column with different superscripts are significantly different from each other (P < 0.05).

reached

a plateau

on day 3. In contrast,

no effect on the alkaline studies showed

phosphatase

that the increase

due to an increase

in V,,,

phlorizin activity.

in enzyme

BBMV,

had

Kinetic

activity

was

and not due to a change

dase activity.

phloretin

Sucrase,

did not increase

isomaltase,

and maltase

differente

of enzyme

activity

not produce

activi-

diet group

0.4 and 127.6 f 9.3 (mU/mg

were 16.9 + 1.8, 5.5 +

protein,

any color product

mean -t SE, n =

tivity

segments

Table 2. Time Course of Changes Day

Sucrase (mU/mg protein)

0 1

12.2 f 0.7a 17.7 + l.lb

2 3 6

25.2 26.4 31.7

f 1.7’ k 1.8’ + 2.0d

effect

of phlorizin

on

in Jejunal Disaccharidase

with was no

the control

and

in the standard

glucose

isolated

along

this axis from jejunal

of rats fed the LS and

LS-phlorizin

diets.

Gradients of sucrase and alkaline phosphatase activity along the villus-crypt axis were observed, as reported previously. ‘9*20A significant increase in sucrase activity occurred in the upper and centra1 regions of the villus-crypt axis, when rats were fed phlorizin, al-

activity was only observed when phlorizin was added to diets containing starch (the LS and HS diets) and not when it was added to the CHO-free diet. direct

There

To investigate where the increase in sucrase acoccurred along the villus-crypt axis, enterocytes

were successively

the

incubated

reaction.

The effect of changes in the dietary starch content on the induction of disaccharidase activity by phlorizin is shown in Figure 1. An increase in disaccharidase

determine

wt/wt). Data are

BBMV (0.32 f 0.04 and 0.27 -t 0.03 n = 3). In addition, phlorizin itself did

6), respectively.

To

between

0.1 lb 0.1 lb o.ogc 0.19’ o.44d

Distribution of Sucrase Activity Along the Villus-Crypt Axis

ties in the LS diet group were 15.2 f 1.2,3.8 -+ 0.3 and 133.8 + 10.6 (mU/mg protein, mean i SE, n = 5), and in the phloretin

in BBMV

was determined.

oxidase

disacchari-

activity

for 1 hour

phlorizin-treated U/mg protein;

in

K,,, (Table 3). Because phlorizin consists of phloretin and glucose, the effect of phloretin was also examined. A diet containing

the sucrase

phlorizin

f O.Ola f O.Olb 0.01” f O.OIC + O.Old * 0.04e

though

there was no differente

Activity in Rats Fed a Phlorizin-Containing

Isomaltase (mU/mg protein)

Maltase (mU/mg protein)

in the sucrase

activity

Diet ALK-Pase (U/mg protein)

3.2 f 0.3”

106.5 f 8.8”

1.2 + 0.1

5.5 6.6 7.3 8.5

149.2 191.4 197.8 217.6

1.320.1 1.4 Ik 0.1 1.6 +I 0.1 1.6 f 0.2

+ ? + k

o.5b 0.2b,c 0.7Cf@ 0.2“

+ f f f

9.8’ 6.7” 12.3” 8.8’

NOTE. After receiving an LS diet for 7 days, rats were fed an LS-phlorizin diet (2 mmol phlorizin per 100 g of LS diet) for the indicated periods. Values are expressed as the mean f SE (n = 6). ALK-Pase, alkaline phosphatase. Values in the same column with different superscripts are significantly different from each other (P < 0.05).

September

PHLORIZIN AND INTESTINAL DISACCHARIDASE

1993

Table 3. Kinetic Data for Disaccharidases in Jejunal BBMV of the LS Diet and LS-Phlorizin Diet Groups Sucrase V_ (U/mg protein) LS diet LS-phlorizin diet K, (mmol/L) LS diet LS-phlorizin diet

Isomaltase

Maltase

due to structural

19.5 + 1.1 19.7 + 1.2

0.11 f 0.02 0.21 k 0.03’ 0.32 i 0.10 0.28 + 0.10

2.0 t 0.3 3.4 + o.a= 0.48 + 0.09 0.35 f 0.16

NOTE. Data are expressed as the mean f SE (n = 3). ‘Significant differente between the LS and LS-phlorizin groups (P < 0.01).

modifications

of

the jejunal membrane elicited by the binding of phlorizin to BBMV, sucrase and alkaline phosphatase activities

0.41 f 0.04 0.54 f 0.16’

and/or functional

695

were

measured

after

phlorizin in vitro. Treatment did not affect the activity

incubating

BBMV

of BBMV with phlorizin of membrane-bound

zymes, suggesting that the increase by phlorizin.

en-

in disaccharidase

activity was not due to direct modification membrane

with

In addition,

of jejunal

the finding that

0.08 -

of the lower villus and crypt cells between the LS and

0.06 -

LS-phlorizin diet groups (Figure 2). There was also no differente in the distribution and leve1 of alkaline phosphatase activity between the LS and LS-phlorizin diet groups.

Effect of Phlorizin on the Na+-Dependent Glucose Transporter Na+-dependent

phlorizin

prepared from rats maintained was significantly

greater than the binding

from rats fed an LS diet (Figure were

no differences

binding

to BBMV

shown).

The

B,,

binding

to BBMV

on an LS-phlorizin

to BBMV

3). However,

of Na+-independent

diet there

phlorizin

between the groups (data not of phlorizin binding in the LS-

phlorizin diet group was significantly

Low

High

Free

Starch

Starch

.-

a, 0 0.025 r Isomaltase 2 0.020 -

r 5

0.015 0.010

-

; + .->

0.005

-

*

greater than that

in the LS diet group (40.1 +- 5.2 vs. 62.2 f 7.2 pmol/ mg protein, mean f SE, n = 5, P < O.Ol), aithough there was no differente

CHO

in the I(d values of the two

0-

groups (5.5 f 0.8 vs. 4.4 f 0.8 pmol/L).

Discussion

CHO

Low

High

Free

Starch

Starch

This study showed an increase of jejunal disaccharidase

activity

and an increase

of Na+-dependent

‘*’ 0.5 -

glucose transporter expression in rats fed a diet conIntestinal disaccharidase activity taining phlorizin. and glucose transport are influenced by fasting and by the time of death.2’-23 Addition of phlorizin to the diet

0.4

-

0.3

-

had no effect on food consumption, the growth of the rats, or intestinal weight, suggesting that the increased

0.2

-

Maltase

*

0.1 -

disaccharidase activity was not due to a change of feeding habits. In addition, a diet containing phloretin (constituent of phlorizin) did not increase disaccharidase activity. This indicated that the effect of phlorizin was not mediated by phloretin, which is produced by phlorizin hydrolase 24 in the brush border membrane.

Figure 1. Effect of varying the dietary starch content on the increase

It has been reported that phlorizin binds firmly to the Na+-dependent glucose transporter.‘3,‘4 To examine whether the increased disaccharidase activity was

in disaccharidase activity produced by phlorizin. Rats were maintained for 7 days on carbohydrate-Wee, low-starch, or high-starch diets without phlorizin (0) or with phlorizin (W: 2 mmol/100 gof diet). Values are expressed as the mean f SE (n = 6).

O-Im CHO

Low

High

Free

Starch

Starch

696

MINAMI

0.06

GASTROENTEROLOGY

ET AL.

,

, 1.2

proteinwas21.4f

0.05

1 .-z

c yC 0.04 0 n

0.8

E 0.03 3

0.6

!! z 0.02 z

0.01

0.2

DNA

it was

(mean

-t SE,

caused

20

40

60

80

Villus protein

of sucrase-expressing

that mature

cells of the villus

synthesis duced

showed

in K, suggests

may account

an increase

that promotion

for the increase

in V,,,

activity

of sucrase

gram of DNA also increased containing

diet: sucrase

expressed

but

expressed

2 Phlorizin

4

6

8

concentration @MI

10

stimulation

of disacwith

the villus

Addition

of phlorizin

a

observed

diet (Figure

and cause an increase

centration

in the

intestinal

it it

1). This finding for the

due to phlorizin.

to the diet should

absorption

when

and not when

mechanism

activity

and

column.6

starch

possible

in disaccharidase

sorption,

per milli-

inhibit

glucose

in the glucose

lumen.

Despite

in the LS diet, the glucose

and this may have triggered

Another

new finding

zin ingestion

induced

phlorizin

as mU/mg

0

of

is in agreement

was only

con-

the low

concentra-

the induction

of

much

binding

greater

of this study was that phlorian increase

to BBMV.

affinity

10

20

30

40

50

Bound (pmol/mg

protein)

60

70

in Na+-dependent

Because

for the glucose

I

0

are capable

disaccharidases.

pro-

in the rats fed a phlorizin-

activity

column

tion very close to the apical surface of the enterocytes may have been increased by inhibition of glucose ab-

of enzyme

in activity

the following

increase

content

of the

of both sucrase-isomaltase along

to diets containing

suggested

of extension

cells. It also suggests

the effect of a sucrose-rich

was added to a CHO-free

by phlorizin.

The specific

result

The effect of phlorizin

starch

no a change

Our

study investigating

was added

isolated

occurred

in the leve1 of expres-

to phlorizin-induced activity.

maltase-glucoamylase

Figure 2. Distribution of sucrase (0) and alkaline phosphatase (a; ALK-Pase) activity in successively collected villus cel1 fractions. Data points represent the mean ? SE (n = 4) for low-starch diet (0) and phlorizin-containing (0) groups. The abscissa shows the percentage of the total protein content. *Significant differente between the two groups (P < 0.01).

al1 the disaccharidases

of an increase

diet on the distribution

100

in sucrase

of phlorizin

distribution

charidase

Crypt

% of total

because

responding

Q

0 0

2, the increase

ingestion

sion per villus cells and not because

previous 0

in Figure by

in the villus cells. This suggests that the activity

increased

Y

respectively,

0.46 + 0.05 and 1.60 t- 0.06, respectively

mainly

j

0

diet groups,

as units per milligram

activity

: 1

0.4

expressed

n = 4). As shown

g a

No. 3

1.5and60.2f8.7(meankSE,n=

4) in the LS and LS-phlorizin although

Vol. 105,

phlorizin carrier

than

has a glu-

Figure 3. (A) Na+-dependent phlorizin binding to jejunal BBMV from rats maintained on the low-starch diet (0) and phlorizin-containing low-starch diet (0) for 7 days. Results are expressed as the mean + SE for 5 rats. Na+-dependent phlorizin binding was determined by subtracting the amount of phlorizin bound in the presence of KSCN from that in the presence of NaSCN. There was a significant difference of Na+-dependent phlorizin binding between the two groups of rats (*P < 0.01). (E?) Scatchard plot of phlorizin binding.

September

1993

PHLORIZIN AND INTESTINAL DISACCHARIDASE

case itself, its binding transporter larity

density

of the

groups

can serve a measure

in intestinal

enrichment

of sucrase

of rats indicates

binding addition,

because phlorizin

binding

of

phlorizin.

glucose

suggested

dietary

crease

above,

the glucose

of enterocytes.

crease

of the glucose

ported previously the jejunal glucose high-carbohydrate drate-free

in

transporter intake This

the re-

12.

close

transporter,

bind-

number

expression.

may then

of in

diet than

should

in-

14.

to the apical trigger

because

in those given addition

13.

As

an in-

it was re-

that specific binding of phlorizin to transporter was greater in mice fed a

diet. 26 In conclusion,

to the diet increased and Na+-dependent

11.

an increase

of phlorizin

concentration

surface

In

in the nonspecific

increase

10.

in Na+-inde-

the groups,

sites may represent

Na+-dependent

al1

in purification.

between

The

phlorizin-binding

in

in phlorizin

there was no differente

sult was not due to an increase ing

activity

that the increase

was not due to variations

pendent

of glucose

mucosa.‘4*25 The simi-

15.

a carbohyof phlorizin

the jejunal disaccharidase activity glucose transporter expression.

16.

The trigger for these changes might have been an increase in the luminal glucose concentration close to

17.

the apical

la.

surface

of the enterocytes.

References

19.

1. Deren JJ, Broitman SA, Zamcheck N. Effect ofdiet upon intestinal

2.

3.

4.

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Received January 24, 1992. Accepted April 26, 1993. Address requests for reprlnts to: Hisanori Minaml, Ph.D., Department of Nutrition, School of Medlclne, The Unlverslty of Tokushlma, Kuramoto-cho, Tokushima 770, Japan. The authors thank Professor Eiji Takeda for hls encouragement dwing this study.