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Department of Nutrition, School of Food and Nutritional Sciences, The University of Shizuoka, 52–1 Yada, Shizuoka 422-8526, Japan
2To whom correspondence should be addressed.
 |
ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-methylglucoside generally did not
increase the transcript levels of SI or the intestinal hexose
transporters. Nuclear run-on assays revealed that fructose as well
as sucrose increased the transcription of both SI and GLUT5 genes and
that the transcription rates of these genes were unaffected by glucose.
These results suggest that fructose (or a metabolite) is capable of
increasing the mRNA levels of SI and hexose transporters in the small
intestine and that transcriptional regulation might play a pivotal role
in the carbohydrate-induced coordinate enhancement of SI and
fructose transporter gene expression
KEY WORDS: • sucrase-isomaltase • gene expression • fructose • hexose transporters • rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Thorens 1996
).
Sucrose hydrolyzed by the action of sucrase-isomaltase
(SI)3
(Hunziker et al. 1986
) produces glucose and fructose,
which are transferred across the brush-border membranes by the
action of sodium-dependent glucose transporter (SGLT1)
(Hediger and Rhoads 1994
) and fructose transporter
(GLUT5) (Davidson et al. 1992
, Rand et al. 1993
), respectively. The liver-type facilitative glucose
transporter (GLUT2) is expressed in the basolateral membranes of
intestinal absorptive cells, where GLUT2 is capable of transferring
glucose, fructose, or galactose from absorptive cells to the blood
stream (Thorens et al. 1990
). We reported previously
that dietary sucrose enhanced both SI mRNA and SGLT1 mRNA levels in the
rat jejunum within 12 h (Yasutake et al. 1995
).
Similarly, Miyamoto et al. (1993)
demonstrated that the mRNA levels of
jejunal hexose transporters (SGLT1, GLUT5, and GLUT2) were elevated by
feeding a fructose diet to rats for 5 d. These studies prompted us
to hypothesize that certain types of monosaccharides or its metabolites
should be capable of coordinately enhancing the gene expression of both
digestive enzymes (e.g. sucrase-isomaltase) and hexose transporters in
the rat small intestine. |
MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Twenty-eight 7-wk-old male Sprague-Dawley rats (Japan SLC,
Hamamatsu, Japan) were fed a low-carbohydrate, high-fat
diet4
(Goda et al. 1995
) for 7 d. They were subsequently
force-fed the low-carbohydrate, high-fat diet or liquid diets
containing glucose, fructose, sucrose, -methylglucoside, or
glycerol5
three times, over a 12-h period, as described previously (Goda and Takase 1994
, Goda et al. 1984
). The amount
of diet given was 2.9 mL per feeding, providing 83.4 kJ/100g body
weight per 12 h. Our previous study, using the same feeding
protocol, showed that sucrase activity exhibited a dose-dependent
response to sucrose between 20 and 70% energy levels and that a dose
of 40% of the energy as carbohydrate was sufficient to detect the
effect consistently (Goda et al. 1985
). The rats were
killed by decapitation between 10:00 and 11:00 h. To examine whether a
decrease in the fat content of the carbohydrate diets affected sucrase
activity and SI mRNA levels, another group of rats was force-fed a
diet identical to the low-carbohydrate diet except that the amount
of fat was reduced to the level of the high-carbohydrate
diets(low-fat diet). The experiment was repeated with selected
dietary groups (control, glucose, fructose, and sucrose) for nuclear
run-on assays. The experimental procedures used in this study met
the guidelines of the animal use committee of the University of
Shizuoka.
Preparation of intestinal samples.
A 1.0-cm segment (100 mg) was excised from the middle region of the jejunal segment and immediately used for RNA extraction. An adjacent 1.0-cm segment was excised and quickly frozen in liquid nitrogen. This second segment was homogenized in 0.5 mL ice-cold 50 mmol sodium phosphate buffer/L (pH 7.0). Aliquots of the homogenate were stored at -20°C for the assay of sucrase activity. The mucosa was scraped from the remaining part of the jejunal segment with a glass microscope slide and immediately subjected to nuclei preparation.
Enzyme assays.
Sucrase activity was assayed as described by Dahlqvist (1964)
with 28
mmol sucrose/L as substrate. Protein content was determined
according to the method of Lowry et al. (1951)
.
RNA extraction and Northern blot hybridization.
Total RNA was extracted as described by Chomczynski and Sacchi (1987)
.
For Northern blot analysis, 20 µg of total RNA per lane was used as
described previously (Yasutake et al. 1995
).
Hybridization was performed using 32P-labeled rat SI cDNA,
rat SGLT1 cDNA, rat GLUT5 cDNA, and rat GLUT2 cDNA as described
previously (Yasutake et al. 1995
). The cDNA probes were
labeled with [-32P] dCTP (111 TBq/mmol, ICN
Biochemicals, Costa Mesa, CA) using a random primer DNA labeling system
(Takara Shuzo, Kyoto, Japan). The radioactivity retained on the
membranes was analyzed with the image analyzer (BAS 2000, Fuji Film,
Tokyo, Japan). Control hybridization was carried out using a rat
ß-actin cDNA.
The rat SGLT1, GLUT5, and GLUT2 cDNA clones were generous gifts from Dr. K. Miyamoto (Tokushima University).
Isolation of intestinal nuclei and nuclear run-on assays.
Nuclei were isolated from jejunal mucosa, and nuclear run-on assays
were carried out by a modification (Tanaka et al. 1998
)
of the method described by Traber et al. (1990)
and Krasinski et al. (1994)
. The numbers of jejunal nuclei were adjusted to 5 x
107 for each assay. [-32P] UTP (111
TBq/mmol, 3.7 TBq) was used for transcription reactions. The
determination of radioactivity retained on the membranes and the SI
cDNA, GLUT5 cDNA, and ß-actin cDNA probes used for run-on assays
were the same as used for Northern blot hybridization.
Statistical analysis.
All results were subjected to one-way ANOVA. Differences in mean values among groups were tested using Tukey's multiple range test and were considered different at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Sucrase activity in the jejunal homogenate of rats force-fed
glucose, fructose, sucrose, -methylglucoside, or glycerol were 157,
218, 201, 88, and 126% greater than that of controls, respectively
(Table 1
). In contrast, feeding a low-carbohydrate, low-fat diet did
not affect sucrase activity; an apparent increase (31%) was caused by
the decrease in the total protein in the jejunum due to reduced energy
intake (data not shown).
|
The levels of SI mRNA were significantly elevated in rats force-fed
a diet containing fructose (242%), sucrose (270%), or glycerol
(192%) compared to controls (Fig. 1
). By contrast, the SI mRNA levels of rats fed a diet containing
glucose or -methylglucoside or the low-carbohydrate, low-fat
diet were not different from those of controls. Thus, the increases in
sucrase activity induced by various carbohydrates, other than glucose
and -methylglucoside, occurred in parallel with the rise in SI mRNA
levels. The levels of SGLT1 mRNA were significantly elevated in rats
given a diet containing glucose (53%), fructose (89%), sucrose
(77%), or glycerol (74%) compared to controls (Fig. 1)
. The SGLT1
mRNA level was unaffected by feeding the low-carbohydrate, low-fat
diet or the -methylglucoside diet (Fig. 1)
. The levels of GLUT5 mRNA
were significantly elevated in rats given a diet containing fructose
(149%) or sucrose (120%) compared to controls (Fig. 1)
, but its
levels were unaffected by any of the other diets tested. The levels of
GLUT2 mRNA were significantly elevated in rats force-fed a diet
containing fructose (169%), sucrose (222%), or glycerol (124%)
compared to controls (Fig. 1)
. The GLUT2 mRNA levels were unaffected by
feeding the low-carbohydrate, low-fat diet; the glucose diet; or
the -methylglucoside diet (Fig. 1)
.
|
When normalized to ß-actin signals, the transcription rates of the SI
gene in rats fed the fructose or sucrose diet were 135 and 81% greater
than that of controls, respectively (Fig. 2
). The transcription rate of the GLUT5 gene in rats fed the
fructose or sucrose diet were 128 and 69% greater than that of
controls, respectively. By contrast, feeding the glucose diet did not
affect the transcription rates of the SI and GLUT5 genes.
|
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) as well as SI mRNA levels
(Broyart et al. 1990
, Yasutake et al. 1995
) in rat small intestines. These results are also
compatible with the work of Miyamoto et al. (1993)
who showed that
feeding a diet rich in fructose to rats caused a coordinate rise in the
mRNA levels of the intestinal hexose transporters, including SGLT1 and
GLUT5. In this study using nuclear run-on assays, we further
demonstrated that the diet-induced elevation of SI mRNA levels was
attributable to the rise in the transcriptional rate of the SI gene. We
recently demonstrated that feeding a fructose diet to rats enhanced the
transcription of lactase-phlorizin hydrolase as well as GLUT5
(Tanaka et al. 1998
). These results suggest that dietary
fructose can, in a short period of time, enhance the expression of the
genes encoding disaccharidases and the hexose transporter in the
intestinal absorptive cells.
In the present study, we found that neither a nonmetabolizable sugar
(-methylglucoside) nor glucose, was able to enhance the mRNA levels
or the transcription of SI and GLUT5 within 12 h. This result led
us to consider the possibility that glucose was less effective in
stimulating the transcription of SI and the fructose transporter genes.
However, it should be noted that Miyamoto et al. (1993)
demonstrated
that feeding a high-glucose diet to rats for 5 d enhanced
SGLT1 and GLUT2 mRNA levels in the jejunum. We previously observed that
both SI and SGLT1 mRNA levels were elevated in the jejunum of rats fed
a high-starch diet for 7 d compared to those fed a
low-starch diet (Yasutake et al. 1995
). The apparent
contradiction of the results concerning the ability of dietary glucose
to increase SI and SGLT1 mRNA accumulation might be explained by
differences in the experimental time periods. Indeed, Miyamoto et al. (1993)
showed that it took 3 d to detect the glucose-induced
increase in SGLT1 mRNA levels. Thus, it is likely that there are at
least two mechanisms involved in the carbohydrate-induced
enhancement of SI and intestinal hexose transporter gene expressions:
one might involve a rapid (and possibly direct) stimulation of gene
transcription by certain metabolizable sugars, including fructose, and
the other mechanism may operate in response to the long-term
consumption of starch or glucose.
We demonstrated in this study that dietary glycerol enhanced SI, SGLT1,
and GLUT2 mRNA levels. We previously showed that the SI and SGLT1 mRNA
levels were significantly enhanced in rats fed a diet rich in
medium-chain triacylglycerols (MCT) compared to those fed a diet
rich in long-chain triacylglycerols (Yasutake et al. 1995
). Because MCT are easily hydrolyzed to medium-chain
fatty acids and glycerol in the small intestine, the MCT-induced
enhancement of the SI and hexose transporter mRNA levels should be
attributable to the glycerol produced from MCT. We contend that a
glycerol metabolite in the absorptive cells may be involved in the
elevation of these mRNA levels. It is unclear at present, however, why
glycerol did not alter the GLUT5 mRNA level. Because feeding a
high-glucose diet to rats for 5 d did not enhance the GLUT5
transcription rate (Miyamoto et al. 1993
), it is
possible that the regulation of GLUT5 gene expression is strictly
mediated by fructose and/or its metabolite(s).
SI is the only enzyme in the small intestine that can hydrolyze
sucrose. Therefore, it seems reasonable that sucrose or its
constituting monosaccharides should play a pivotal role in the
regulation of SI gene expression. Sucrose is hydrolyzed to glucose and
fructose, and these two monosaccharides are subjected to
carrier-mediated transfers through the brush-border membrane by
SGLT1 (Wright 1993
) and by GLUT5 (Burant et al. 1992
), respectively. In this study, we found that fructose
enhanced the SI and intestinal hexose transporter mRNA levels within
12 h, which is consistent with recent reports that showed that
dietary fructose caused a rapid increase in GLUT5 mRNA levels in the
jejunum of adult rats (Corpe and Burant 1996
)
and in weaning rats (Shu et al. 1997
). Corpe et al. (1998)
demonstrated that after 4 h of fructose feeding, GLUT5 mRNA
and protein levels increased 2–3.5–fold above the basal levels of
expression. It was also reported, when using the human colon carcinoma
cell line Caco-2, that the addition of fructose to the medium elevated
the GLUT5 mRNA level in as little as 8 h (Mesonero et al. 1995
). Taken together, it is likely that fructose not only
enhances the transcription of various genes related to intestinal
carbohydrate digestion and absorption, but also induces its own
transporter. This may result in a positive feedback of fructose
transport and a pronounced effect of fructose on SI and GLUT5 gene
expression.
In conclusion, the present study demonstrated that the levels of SI and GLUT5 mRNA were concomitantly elevated by dietary fructose, but not by dietary glucose. This suggests that fructose or its metabolites induce an increased transcription of SI and the GLUT5 transporter, probably through a common regulatory mechanism. Further studies are required to explore whether carbohydrate response elements are present in the SI and the fructose transporter genes and what sorts of transcriptional factors participate in this sugar-mediated transcriptional control.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: GLUT2, glucose transporter
type 2; GLUT5, glucose transporter type 5; MCT, medium-chain
triacylglycerols; SGLT1, sodium/D-glucose transporter 1; SI,
sucrase-isomaltase.
4 Composition of diet (g/kg diet): casein (159),
cornstarch (36), corn oil (247), AIN-76 mineral mix (AIN 1977)
(28), AIN-76 vitamin mix (AIN 1977
) (8), DL-methionine
(2.4), choline bitartrate (1.6), 2 g/L agar (518).
5 The diets contained 40% of energy or equivalent
weight as carbohydrate. Composition (g/L diet): casein (128),
carbohydrate (237), corn oil (96.2), AIN-76 mineral mix (21.5),
AIN-76 vitamin mix (6.3), DL-methionine (1.8), choline
bitartrate (1.2).
Manuscript received September 18, 1998. Revision accepted January 26, 1999.
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