Báo cáo Y học: Expression of glucose transporter-2, glucokinase and mitochondrial glycerolphosphate dehydrogenase in pancreatic islets during rat ontogenesis potx

Expression of glucose transporter-2, glucokinase and mitochondrial
glycerolphosphate dehydrogenase in pancreatic islets during rat
ontogenesis
Marta Garcõ
Â
a-Flores
1
, Jose
Â
Antonio Zueco
1
, Joaquõ
Â
n Arenas
2
and Enrique Bla
Â
zquez
1
1
Department of Biochemistry and Molecular Biology, Faculty of Medicine, Complutense University, Madrid, Spain;
2
Clinical Biochemistry Service, Ô12 de OctubreÕ Hospital, Madrid, Spain
To gain better i nsight into the insulin secretory activity of
fetal bcells in response t o glucose, the expression of glucose
transporter 2 (GLUT-2), glucokinase and mitochondrial
glycerol phosphate dehydrogenase (mGDH) were studied.
Expression of GLUT-2 mRNA and protein in pancreatic
islets and liver was signi®cantly lower in fetal and suckling
rats than in adult rats. The glucokinase content of fetal islets
was signi®cantly higher than of suckling and adult rats, and

in liver the e nzyme appeared for the ®rst time on about day
20 of extrauterine life. The highest content of hexokinase I
was found in fetal islets, after which it decreased progres-
sively to the adult values. Gluco kinase mRNA was abun-
dantly expressed in the islets of all the experimental groups,
whereas in liver it was only present in adults and 20-day-old
suckling rats. In fetal islets, GLUT-2 and glucokinase pro-
tein and their mRNA increased as a function of increasing
glucose concentration, whereas reduced mitochondrial
citrate synthase, succinate dehydrogenase and cytochrome c
oxidase activities and mGDH expression were observed.
These ®ndings, together with those reported b y others, may
help to explain the decreased insulin secretory activity of fetal
b cells in response to gluc ose.
Keywords: glucokinase; GLUT-2; mitochondrial g lycerol-3-
phosphate dehydrogenase; ontogeny; pancreatic islets.
In many mammals the ability of pancreatic b cells to
secrete insulin in response to glucose appears after birth
[1], and even the decreased glucose tolerance process
endures for the whole suckling period [2]. It is condi-
tioned by development and environmental events, such as
the hormonal and nutritional changes that characterize
the ontogenic period [3,4]. Although several explanations
have been offered for the unresponsiveness of fetal
pancreatic bcells to glucose, the cellular mechanisms
involved in this process are not well understood. The
delayed appearance of glucose transporter-2 isoform
(GLUT-2) and glucokinase as components o f a
glucose-sensing system [5] in pancreatic b cells during
development could explain such glucose insensitivity.

However, the presence of GLUT-2 and glucokinase
reported by us here, and by other authors [6,7], suggests
that the inability of fetal p ancreatic b cells to secrete
insulin in response to glucose may not be due to the lack
of the glucose-sensor system. However, the situation may
be more complex because of differences in transcription
and translation of GLUT-2 and glucokinase genes. In
fact, the immature secretory response to glucose in
neonatal pancreas may be related to de®cient glucokinase
as well as to reduced GLUT-2 gene expression [8]. Also,
during the fetal period, the post-translational control of
pancreatic glucokinase by glucose may not necessarily be
present, as happens in adult animals [9]. In the light of
the above, we were prompted to study the effect of
different glucose concentrations on the expression of both
GLUT-2 and glucokinase mRNA and protein from fetal
pancreatic islets.
Because the newborn rat is relatively immature, the
number and oxidative activities of mitochondria at this
stage may be reduced and consequently produce
decreased amounts of ATP, which would in turn affect
the ATP-sensitive K
+
channels and, ®nally, the secretion
of insulin. Bearing this in mind, we determined the
activities of citrate synthase (an enzyme of the Krebs
cycle re¯ecting the number of mitochondria [10]), succi-
nate dehydrogenase and cytochrome oxidase (compo-
nents of the respiratory chain) during rat ontogeny.
Mitochondrial glycerol-3-phosphate dehydrogenase

(mGDH) forms part of the shuttle that transports the
NADH generated i n g lycolysis into mitochondria
through an oxidation±reduction cycle which results in
generation of FADH
2
, which is oxidized in the electron-
transport chain. mGDH activity in pancreatic islets is
much higher than in other tissues [11] and is involved in
insulin secretion. Accordingly, we studied the expression
of this enzyme in rat pancreatic islets during develop-
ment, comparing the results obtained with t hose found
for the above variables.
Correspondence to E. Bla
Â
zquez, Departamento de Bioquõ
Â
mica,
Facultad de Medicina, Universidad Complutense, 28040-Madrid,
Spain. Fax: 34 91 3941691, Tel.: 34 91 3941443,
E-mail:
Abbreviations: GLUT-2, glucose transporter-2; mGDH,
mitochondrial glycerol-3-phosphate dehydrogenase; GST,
glutathione S-transferase.
(Received 2 August 2001, revised 9 October 2001,
accepted 23 October 2001)
Eur. J. Biochem. 269, 119±127 (2002) Ó FEBS 2002
MATERIALS AND METHODS
Experimental animals
Wistar rats were housed under constant light (lights on
0800±2000 h) and temperature, with free access to food

and water. Female rats, weighing 200±225 g, were caged
with males until mating had occurred. Vaginal smears
were examined daily for spermatozoa early each
morning. Pregnancy was dated from the ®rst day on
which spermatozoa were identi®ed. The accuracy of this
method of dating was estimated to have a 6±12 h error.
All procedures were carried out according to European
Community ethical regulations for animal research.
Preparation of pancreatic islets
Islets were isolated from the pancreas of adult male rats
(200±225 g) by the procedure of Lacy & Kostianovsky
[12] as modi®ed by Gotoh [13], using collagenase
P (1±1.6 mgámL
)1
) and DNase I (1 mgámL
)1
). Pancre atic
islets from fetuses and 5, 10, and 20-day-old suckling rats
were isolated. Immediately after the animals had been
killed, their pancreases were removed and cut into small
pieces. The fragments were transferred to vials containing
10 m
M
Hanks/Hepes buffer, pH 7, DNase I (1 mgámL
)1
),
and collagenase P (1.6, 1.7, 1.8 and 2 mgámL
)1
for
21-day fetuses and 5, 10, and 20-day-old suckling rats,

respectively), and i ncubated at 37 °C for 10±12 min.
Islets from all the experimental groups were separated
from exocrine tissue by Ficoll gradient centrifugation
[14].
Insulin secretion by pancreatic islets
Samples of 10 islets, obtained from fetal, suckling and adult
rats as described above, were transferred to ¯asks contain-
ing 2 mL Krebs/Ringer bicarbonate, pH 7.2, e nriched with
1% BSA and glucose (1.67, 5.5 or 16.7 m
M
), and placed in a
metabolic incubator in an atmosphere of 95% O
2
and 5%
CO
2
at 37 °C. After 2 h of incubation, the islets were
removed by centrifugation at 280 g for 5 min, and the
supernatants obtained were frozen at )80 °C until the
insulin assays. Insulin was measured by the radioimmun o-
assay method of Herbert et al. [15], using rat insulin as a
standa rd.
RNA isolation and Northern-blot analysis
Total cellular RNA from homogenates of fresh or
cultured pancreatic islets and liver extracts from 21-day
fetuses, 5, 10 and 20-day-old suckling and adult rats was
isolated by the a cid/guanidinium isothiocyanate method
[16]. Total RNA was size-fractionated t hrough a 1.3%
agarose/formaldehyde gel an d transferred to a nylon
membrane. Blots were probed under high-stringency

conditions with an antisense digoxigenin-labelled cRNA
probe generated with SP6 RNA polymerase in p GEM7
for the cDNA of GLUT-2, generously donated by
B. Thorens, Lausanne, Switzerland, using the DIG
RNA labelling kit (Boehringer-Mannheim, Germany).
CDNA synthesis, PCR ampli®cations, and Southern-blot
analysis
Using random primers, the ®rst-strand glucokinase cDNA
was prepared from total RNA isolated from pan creatic
islets and liver extracts of fetal, suckling and adult rats, using
the reverse transcription system for ®rst-strand cDNA
synthesis (Promega). Oligonucleotide primers correspond-
ing to nucleotide [17] bases 821±840 (5¢-CCACATTCTG
CATTTCCTC-3¢) and 276±296 (5¢-GTCTAAAGATGT
TACCCACC-3¢)weredesignedtoamplifya564-bpfrag-
ment of the coding region of rat glucokinase cDNA. PCR
ampli®cation was carried out using an annealing tempera-
ture of 58 °C, except for the ®rst ®ve cycles at 62 °C, and an
extension temperature of 72 °C for 30 cycles. To control for
differences in initial RNA levels and tube-to-tube
variations in RT-PCR, a primer pair for 18S rRNA that
gives rise to a 488-bp cDNA product was included in e ach
PCR ampli®cation. The ampli®cation products were size-
fractionated in 5% polyacrylamide gel and transferred to a
nylon membrane. Blots were hybridized under high-strin-
gency conditions with glucokinase, and 18S RNA probes
were labelled with digoxigenin using the DIG-RNA label-
ling kit.
Western-blot analysis
GLUT-2, glucokinase, and mGDH proteins were identi®ed

by Western blots. Depending on the experimental group,
100±300 fresh or cultured pancreatic islets were sonicated
for3sat4°C in 100 lL lysis buffer containing 5% SDS,
80 m
M
Tris/HCl, pH 6.8, 5 m
M
EDTA, 10% glycerol and
1m
M
phenylmethanesulfonyl ¯uoride. The lysates were
centrifuged, and the protein contents o f the resulting
supernatants were measured by the Bio-Rad protein assay
kit. Liver pieces were homogenized in buffer containing
1m
M
Tris/HCl, pH 7.5, 1 m
M
MgCl
2
,10l
M
phenyl-
methanesulfonyl ¯uoride, 1 lgálL
)1
leupeptin and 3 m
M
dithiothreitol. Aliquots of the homogenates were used to
determine protein content. Samples from pancreatic islets
and liver extracts were resolved by electrophoresis through

an SDS/polyacrylamide gel (10%) and electrotransferred on
to nitrocellulose ®lters. After being blocked in Tris-buffered
saline (20 m
M
Tris/HCl, pH 7.4, 150 m
M
NaCl) containing
0.2% Nonidet P40 and 5% nonfat dry milk overnight at
4 °C, the ®lters were incubated with a polyclonal rabbit
antiserum (1 : 2700) against GLUT-2 (East Acres, South-
bridge, MA, USA), a sheep antiserum (1 : 2000) against
glutathione S-transferase±glucokinase (GST±glucokinase)
fusion protein (a gift from M. A. Magnuson, Vanderbilt
University, TN, USA), or a polyclonal rabbit antiserum
against GST±mGDH (a gift from R. Gomis, Hospital
Clinic Barcelona, Spain) for 1 h at room temperature. After
excess antibody had been washed off, the ®lters were
reblocked in Tris-buffered saline containing 5% nonfat dry
milk and 0.2% Nonidet P40 for 60 min at room temper-
ature and incubated with an anti-rabbit IgG or an anti-
sheep IgG con jugated to horseradish peroxidase for 1 h at
room temperature. Chemiluminescence detection was car-
ried out in the presence of ECL reagents from the
Radiochemical Centre, Amersham, Bucks, UK.
120 M. Garcõ
Â
a-Flores et al. (Eur. J. Biochem. 269) Ó FEBS 2002
Determination of mitochondrial enzyme activities
As a ®rst step to the determination of citrate synthase,
succinate dehydrogenase and cytochrome c oxidase activ-

ities, isolated p ancreatic islets were homogenized in 100 lL
Krebs/Ringer phosphate, pH 7.0: 400 were homogenized
for 21-day fetuses, 300 for 10-day-old suckling rats, and 235
for adult rats. They were then sonicated with 1 0 low-
frequency pulses. Pieces of liver were homogenized
mechanically in 15 vol. Krebs/Ringer phosphate buffer,
pH 7.0.
In the citrate synthase assay [18], 100 lL0.75
M
Tris/
HCl, pH 8.0, was mixed with 100 lL 1% Triton X-100,
100 lL5,5¢-dithiobis(2-nitrobenzoic acid), 50 lL acetyl-
CoA (7 mgámL
)1
)and10lL tissue homogenate, and then
made up to 950 lL with distilled water and incubated at
30 °Cfor2min.A
412
was measured for the last minute in a
Beckman DU-68 spectrophotometer. Then, oxalacetate was
added to initiate the b iochemical reaction, and rates of
absorbance were recorded for 2±3 min. To calculate t he
enzyme activity, we subtracted the change in A
412
before the
addition of oxalacetate (which is acetyl-CoA hydrolase
activity) from the change in A
412
after addition of oxalace-
tate.

Succinate dehydrogenase activity [18] was determined
after mixing 4 0 lL tissue homogenate with 500 lLKrebs/
Ringer phosphate buffer, pH 7.0, containing 100 lL
dichloroindophenol and 50 lL KCN, and then made up
to 900 lLwithwater.Then100lL320m
M
succinate
was added and the D
600
was measured for 5 min at 30 °C.
Cytochrome c oxidase activity [18] was determined a fter
the incubation of 100 lL Krebs/Ringer phosphate buffer,
pH 7.0, 100 lL 1% reduced cytochrome c, and 780 lLof
distilled water for 2 min at 38 °C. The spectrum was
recorded from 500 to 600 nm to ensure that the cyto-
chrome c was fully reduced. After the addition of 20 lL
tissue homogenate, the decrease in D
550
was m easu red for
2min.
Mitochondrial enzyme activities (i.e. citrate synthase,
succinate dehydrogenase, and cytochrome c oxidase) were
expressed as nmolámin
)1
á(mg protein)
)1
. In our hands,
analytical variation coef®cients of mitochondrial enzyme
activities were b elow 10% and recoveries ran ged from 90%
to 120%. The protein contents of the samples were

determined by the method of Lowry et al.[19].
RESULTS
Effect of glucose concentration on insulin release
by pancreatic islets of fetal, suckling and adult rats
Table 1 shows that an increase i n the concentration of
glucose in the incubation medium from 5.5 to 16.7 m
M
produced a signi®cant stimulation of insulin secretion by the
pancreatic islets of suckling and adult rats. This effect was
not observed in pancreatic islets from 21-day-old fetuses,
although, surprisingly a small but signi®cant increase in
insulin release was observed when glucose levels were
increased from 1.67 to 5.5 m
M
. As previously reported by us
[2], the amount of insulin released by pancreatic islets
represented 2.8% and 7.8% of the total content in 5 and
10-day-old rats, respectively, compared with the 14.7%
found in young adult animals.
Ontogenesis of
GLUT-2
and glucokinase mRNA
and protein in rat pancretic islets and liver
As compared with adults, in pancreatic islets of fetal and
suckling rats, expression of GLUT-2 mRNA was greater
than that seen for the protein (Figs 1 and 2). However, in all
cases, the mRNA content was signi®cantly lower in younger
than adult animals.
Western-blot analysis revealed a major 62-kDa band,
which corresponded to the GLUT-2 protein in pancreatic

islets and livers of fetal, suckling, and adult rats (Fig. 2). The
intensity of this band was lower in pancreatic islets from
fetal and suckling rats than in those from adult animals
(Fig. 2 A). In liver, GLUT-2 expression was minimal in
21-day fetuses but increased signi®cantly after birth,
although only up to a level below adult values (Fig. 2B).
GLUT-2 expression in suckling rats was higher in liver than
in pancreatic islets, whereas in fetal liver it was almost
undetectable.
Because glucokinase mRNA is dif®cult to detect in
pancreatic islets by Northern blot, we used the RT-PCR
method to amplify the mRNA obtained from 100±300
pancreatic islets from each experimental group. At the same
time as glucokinase cDNA was being ampli®ed, a 488-bp
fragment corresponding to the sequence of 18S rRNA was
used to normalize the results. As shown in Fig. 3A, the
expression of glucokinase mRNA in pancreatic islets was
almost the same in all experimental groups. In contrast, in
liver, glucokinase mRNA was only present in adults and was
present at lower levels in 20-day-old suckling rats (Fig. 3B).
Also, on Western-blot analysis, a 52-kDa protein corres-
ponding to the glucokinase was identi®ed in pancreatic islets
during rat ontogenesis (Fig. 4). However, the developmen-
tal pattern was signi®cantly different in pancreatic islets and
liver. Surprisingly, the glucokinase content of pancreatic
islets was signi®cantly higher in fetuses than in adult
rats and e ven higher than in s uckling animals (Fig. 4A). In
contrast, in the liver this enzyme appeared for the ®rst time
at the end of the suckling period, and even after 20 days of
extrauterine life the protein content was less than 20% of

that found in adults (Fig. 4B). On Western-blot analysis
of pancreatic islets, a 100-kDa protein, corresponding
to hexokinase I, was identi®ed (Fig. 4C). The highest
content of the protein was found in 21-day-old fetuses and
5-day-old suckling rats, and these values decreased signif-
icantly with age.
Table 1. Eect of dierent glucose concentrations on insulin release by
pancreatic islets of 21-day fetuses (F-21), 5 (S-5), 10 (S-10) and 20-day-
old (S-20) suckling and adult rats. Values are means  SEM (n  7)
and are expressed as pg á(n g DNA)
)1
á2h
)1
.
Insulin release
1.67 m
M
glucose 5.5 m
M
glucose 16.7 m
M
glucose
F-21 34.78  3.40
a
68.5  2.69 77.20  3.68
S-5 29.66  8.50 39.17  8.35 64.53  15.17
S-10 61.17  6.95 77.18  4.85 182.8  29.93
a
S-20 120.54  11.71 127.33  10.72 390.45  30.83
a

Adults 132.46  9.99 141.53  8.72 833.8  55.06
a
a
P < 0.05 compared with the data obtained at 5.5 m
M
glucose.
Ó FEBS 2002 GLUT-2, glucokinase and mGDH during development (Eur. J. Biochem. 269) 121
Effect of glucose concentration on the expression
of
GLUT-2
and glucokinase mRNA and protein
in the pancreatic islets of fetal and adult rats
We studied the e ffect of glucose concentration (2.8, 5.5 and
20 m
M
)onGLUT-2 mRNA expression and protein in islets
from 21-day fetuses. As shown in Fig. 5A, GLUT-2 mRNA
in fetal islets increased when the glucose concentration in the
culture medium was changed from 2.8 to 5.5 or 20 m
M
.
In addition, when fetal islets were preincubated with 2.8 m
M
glucose and then incubated with 20 m
M
glucose, the
expression of GLUT-2 mRNA increased signi®cantly. In
contrast, preincubation of fetal islets with 20 m
M
glucose

which were then incubated with 2.8 m
M
glucose produced
the opposite effect. GLUT-2 mRNA levels in fe tal pancre-
atic islets cultured with 20 m
M
glucose and actinomycin D
Fig. 1. Ontogenesis of GLUT- 2 mRNA in rat pancreatic islets and liver.
Total RNA from pancreatic islets (A) and liver (B) of 21-day-old fetal
(F-21), 5, 10, and 20-day-old suckling ( S-5, S-10, and S-20) and adult
rats were hybridized with speci®c probes for GLUT-2 and 18S rRNA.
At the top of both panels are the bands corresponding to the dierent
experimental groups. Densitometric data express the GLUT-2/18S
rRNA ratio, relative to 100% for adult values. Values are means
 SEM from four independent experiments. *P < 0.05, **P <0.01
compared with adults.
Fig. 2. Ontogenesis of GLUT-2 protein in rat pancreatic islets and liver.
Western-blot an alyses o f GL UT-2 in pancreatic islets (A) and liver (B)
from 21-day fetuses (F-21), 5, 10, and 20-day-old suckling (S-5, S-10,
and S-20) and adult rats. At the top of both panels are the bands
corresponding to the dierent experimental groups. Densitometric
data were calculated as the percentage of adult values. Values are
means  SEM from four ind ependen t experiments. * P <0.001
compared with a dults.
122 M. Garcõ
Â
a-Flores et al. (Eur. J. Biochem. 269) Ó FEBS 2002
were dramatically reduced, whereas the addition of cyclo-
heximide did not change GLUT-2 mRNA levels (Fig. 5B);
these ®ndings indicate that high glucose concentrations act

on the transcription of the GLUT-2 gene and that is not
required for the synthesis of new proteins involved in the
transcription of the gene. Likewise, the GLUT-2 protein
content in fetal islets increased (Fig. 5C) when glucose
concentration i n the culture medium was increased. When
fetal islets were switched from 2.8 to 20 m
M
glucose, the
expression of GLUT-2 increased up to 10-fold. In contrast,
when fetal islets were switched from 20 to 2.8 m
M
glucose,
an 85% reduction in protein c ontent w as observed.
Similarly, an increase in glucose concentration in the culture
medium stimulated the expression of the glucokinase
mRNA and protein of fetal p ancreatic islets (Fig. 6); in
addition, both parameters were modi®ed by sequential
incubation of fetal islets with low-to-high or high-to-low
glucose concentrations.
Measurement of mitochondrial enzyme activities
in pancreatic islets and liver of fetal, suckling
and adult rats
We studied the enzyme activities of two protein complexes
involved in the respiratory chain: succinate dehydrogenase
(complex II) and cytochrome c oxidase (complex IV). A
third enzyme, citrate synthase (a Krebs cycle component), is
considered to be a reliable index of mitochondrial content or
number [10]. As shown in Fig. 7, the activities o f all three
enzymes were always lower in 21-day-old fetuses a nd 10-day-
old suckling rats than in adult animals. These differences

were more marked when enzyme activities were studied in
liver (data not shown). However, the differences for the
mitochondrial enzymes are much less obvious in the different
stages of development than for GLUT-2 and glucokinase.
Expression of the mitochondrial protein mGDH
in pancreatic islets and liver during rat ontogenesis
Using Western blot, we assayed the expression of mGDH in
pancreatic islets and liver during rat ontogenesis in an
attempt to uncover whether developmental changes in this
enzyme can explain the above alterations in insulin secretion
in the fetus. A 72-kDa protein corresponding to mGDH
was identi®ed in all e xperimental g roups, but its tissue
content changed signi®cantly during rat development
(Fig. 8 ). In pancreatic islets, the amount of the protein in
21-day-old fetuses was almost h alf t hat f ound in adult rats,
after which it increased progressively during the suckling
period (Fig. 8A). A similar pattern of development was
found in the liver (Fig. 8B), with a protein c ontent of 10%
in fetal rats and 35% in 10-day-old suckling rats relative to
the values found in adult animals.
DISCUSSION
During adult life, pancreatic b cells are able to recognize
changes in circulating nutrients, mainly glucose, through a
sensor system that facilitate s the metabolism o f glucose in
these cells and then the synthesis and secretion of insulin. In
contrast with these ev ents, fetal pancreatic b cells secrete
insulin poorly in response to increased glucose concentra-
tions in the extracellular s pace [1], even though synthesis of
this hormone is stimulated by glucose [2,20].
Our results indicate that GLUT-2 and glucokinase in

b cells are already present during intrauterine life, and data
from the literature con®rm that the glucokinase gene is
Fig. 3. Ontogenesis of glucokinase mRNA in rat pancreatic islets and
liver. Total RNA from pancreatic islets (A) and liver (B) of 21-day-old
fetal (F-21), 5, 10, and 20-day-old suckling (S-5, S-10, and S-20) and
adult rats were hybridized with speci®c probes for glucokinase and 18S
rRNA. At t he top of b oth panels are the bands corresponding to the
dierent experimental groups. Densitometric data express the
GLUT-2/18S rRNA ratio, relative to 100% for adult values. Values are
means  SEM from four independent experiments. *P < 0.05,
**P < 0.01 compared with a dults.
Ó FEBS 2002 GLUT-2, glucokinase and mGDH during development (Eur. J. Biochem. 269) 123
expressed much later in liver than in pan creatic islets. This
may be explained by the presence of tissue-speci®c promo-
ters which allow differential regulation [21±23]. Glucokinase
levels in b cells appear to be c ontrolled by glucose [24],
whereas insulin appears to be the major positive effector of
glucokinase activity i n liver [23,24]. We also found that, i n
fetal pancreatic islets, glucose is able to stimulate the expres-
sion of mRNA and protein corresponding to GLUT-2
and glucokinase. Interestingly, other authors have found
that, in fetal islet b and a cells of the rat [7] and in human
fetal islet-like cell clusters [25], glucokinase activity increased
linearly with increasing glucose concentration. All these
®ndings indicate that the poor release of insulin by fetal
pancreatic b cells in response to glucose [1] is not related to a
lack of GLUT-2 or glucokinase or to the absence of the
induction of these molecules by glucose, despite the fact that
a de®cient glucokinase and reduced GLUT-2 expression
[8,26] have been reported in neonatal pancreas. Acco rdingly,

the lower expression of GLUT-2 also found by us in fetal
pancreatic islets may contribute, at least in part, to the poor
insulin release of b cells in response to glucose. Thus, in
animal models of diabetes the expression of GLUT-2
mRNA and protein is modi®ed [27,28] whereas expression
of GLUT-2 antisense RNA in b cells of transgenic mice
leads to diabetes [29]. In addition, a mutation in the GLUT-2
gene has been reported in a type-2 diabetic patient [30].
Fig. 5. Eect of glucose concentrations on the expression of GLUT-2 mRNA and protein in pancreatic islets of 21-day-old fetuses. (A) Fetal pancreatic
islets were cultured with dierent glucose concentrations (2.8, 5.5 and 20 m
M
) for 48 h. In some cases, pancreatic islets were cultured with either 2.8
or 20 m
M
glucose for 48 h and then shifted to 20 or 2.8 m
M
glucose for 48 h. (B) Fetal pancreatic islets were cultured with die rent glucose
concentrations (2.8, 5.5 and 20 m
M
)or20m
M
glucose and either actinomycin D (10 lgámL
)1
) or cycloheximide (5 l
M
) for 16 h. Total RNA from
fetal pancreatic islets was hybridized with sp eci®c probes for GLUT-2 and 18S rRNA. Densitometric data express the GLUT-2/18S rRNA ratio as
percentage of the values obtained at 5.5 m
M
glucose (A and B ). (C) Western-blot analysis o f GLUT-2 in fetal pancreatic islets cultured with dierent

glucose concentrations (2.8, 5.5 and 20 m
M
) for 16 h. Densitometric data were calculated as the percentage of the values obtained at 5.5 m
M
glucose. Values are m eans  SEM from three or four independent experiments. *P < 0.05, **P < 0.001 compared with 5.5 m
M
glucose.
Fig. 4. Ontogenesis of hexokinase I and glucokinase protein in rat pancreatic islets and liver. Western-blot analyses of glucokinase in pancreatic islets
(A) and liver (B) and hexokinase I in pancreatic islets (C) from 21-day-old fetuses (F-21), 5, 10, and 20-day-old suckling (S-5, S-10, and S-20) and
adult rats. At the top of the th ree panels are the band s corresponding to the dierent e xperimental groups. Densitometric data were calculated as the
percentage of adult rats. Values are m eans  SEM from four independent experiments. *P <0.05,**P < 0.005 compared with adults.
124 M. Garcõ
Â
a-Flores et al. (Eur. J. Biochem. 269) Ó FEBS 2002
We also observed that the highest a mounts of hexokinase I
were present in the pancreatic islets of fetal and 5-day-old
suckling rats, after which its levels gradually decreased
during the nursing period until adult values were reached.
Similar results have been reported by o ther authors in liver,
skeletal muscle and heart [31], although the reduction in
hexokinase I content occurs faster in these tissues than in
pancreatic islets. Because of the low plasma glucose
concentrations (1.6, 1.8 and 4.3 m
M
) in 19, 20 and 21-day
rat fetuses [4], respectively, compared with the 5.5 m
M
concentration in adult animals, hexokinase I, rather than
glucokinase, may be the enzyme responsible for glucose
phosphorylation in fetal pancreatic islets. Interestingly,

neonatal pancreatic b cells start to release insulin in response
to high glucose concentrations when this substrate reaches
normal adult values in the blood circulation [4]. In fact,
continuous glucose administration t o p regnant rats induces
maturation of fetal pancreatic b ce lls so that they release
insulin in response to glucose [32].
Because the rat is born in a n immature state, we studied
the activities of t hree mitochondrial enzymes to obtain
information about the n umber and function of mitochon-
dria during intrauterine life. A lower activity of all three
activities in fetal than adult pancreatic islets, but a similarity
in the index of respiratory enzyme to citrate synthase
Fig. 6. Eect of glucose concentrations on the expression of gluco-
kinase mRNA and protein in pancreatic islets of 21-day fetuses. (A)
Fetal pancreatic islets were cultured with dierent glucose concen-
trations (2.8, 5.5 and 20 m
M
) for 48 h. In some cases, pancreatic islets
were cultured with either 2.8 or 20 m
M
glucose for 48 h and then
shifted to 20 or 2.8 m
M
glucose for 48 h. Total RNA from fetal
pancreatic islets was hybridized with speci®c probes for glu cokinase
and 18S rRNA. Densitometric data express the glucokinase/18S
rRNA ratio as th e percentage of the values obtained at 5.5 m
M
glucose. (B) Western-blot analysis of glucokinase in fetal pancreatic
islets c ultured with dierent glucose concentrations (2.8, 5.5 and

20 m
M
) for 48 h. Densitometric data were calculated as the per-
centage of the va lues obtain ed at 5.5 m
M
glucose. Values are mean-
s  SEM fro m t hree inde pende nt experiments. *P < 0.05,
**P < 0.01 compared with 5.5 m
M
glucose.
Fig. 7. Citrate synthase (CS), succinate dehydrogenase (SDH), and
cytochrome c oxidase (COX) activities in homogenates of pancreatic
islets from fetal, suckling and adults rats. Values are means  SEM
from six independent experiments. *P <0.05, **P < 0.001 com-
paredwithadultvalues.
Ó FEBS 2002 GLUT-2, glucokinase and mGDH during development (Eur. J. Biochem. 269) 125
activity, indicates the presence of fewer mitochondria during
intrauterine life even though the enzyme activities were the
same per mitochondrial unit in fetal, suckling, and adult rats.
Another mitochondrial enzyme i s mGDH, de®ciencies in
the activity or con tents of which have been associated with
type 2 diabetes in humans and experimental animals [33,34].
Interestingly, we observed that expression of this enzyme is
lower in fetal and suckling rats than in adult rats, which,
together with the reduced number of mitochondria and
enzyme activities in the pancreatic islets of younger animals,
may affect glucose-dependent insulin release at this age.
Further support for these ®ndings come from the report of
Welsh et al. [35], who reported that fetal pancreatic islets
have less mRNA for the mitochondrial protein transporter

of adenine nucleotides than adult animals.
The lack of responsiveness of fetal b cells in releasing
insulin in the presence of high g lucose concentrations may
not be due to modi®ed functioning of ATP-dependent K
+
and voltage-dependent Ca
2+
channels because different
insulin secretagogues, but not glucose, increase intracellular
Ca
2+
in fetal rat b cells [36]. H owever, the inability of fetal
b cells to release insulin in response to glucose may be at
least in part due to reduced glucose metabolism [37]
through the pentose phosphate shunt [38], a decreased
number of mitochondria, or the glycerol 3-phosphate
shuttle, which may alter the production of ATP or the
formation of the intracellular signalling messengers re-
quired for stimulation of glucose-dependent insulin secre-
tion. Both maturation of these endogenous processes and
acquisition of adult levels of glucose in the blood at birth
may be needed to render b cells competent to respond to
glucose. In a similar way, we have recently reported [39]
that GLP-1(7±36)amide confers glucose competence on
fetal pancreatic b cells.
ACKNOWLEDGEMENTS
We are indebted to Drs W. J. Malaisse and R. Gomis for critical
reading of the manuscript, and to Mrs Pilar del Hoyo for skillful
technical assistance. Th is investigation was supported by grants f rom
the Direccio

Â
n General de Investigacio
Â
nCientõ
Â
®cayTe
Â
cnica (DGI-
CYT), the Fondo de Investigacio
Â
n Sanitaria de la Seguridad Social, and
the Comunidad de M adrid, Spain.
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