Expression of glucose transporter-2, glucokinase and mitochondrial glycerolphosphate dehydrogenase in pancreatic islets during rat ontogenesis Marta GarcõÂa-Flores1, Jose Antonio Zueco1
Trang 1Expression of glucose transporter-2, glucokinase and mitochondrial glycerolphosphate dehydrogenase in pancreatic islets during rat
ontogenesis
Marta GarcõÂa-Flores1, Jose Antonio Zueco1, JoaquõÂn Arenas2and Enrique BlaÂzquez1
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 insight into the insulin secretory activity of
fetal b cells in response to 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 enzyme 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 Glucokinase 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 by others, may help to explain the decreased insulin secretory activity of fetal
b cells in response to glucose
Keywords: glucokinase; GLUT-2; mitochondrial glycerol-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 b cells 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 of 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 pancreatic 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 in glycolysis into mitochondria through an oxidation±reduction cycle which results in generation of FADH2, 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 those found for the above variables
Correspondence to E BlaÂzquez, Departamento de BioquõÂmica,
Facultad de Medicina, Universidad Complutense, 28040-Madrid,
Spain Fax: 34 91 3941691, Tel.: 34 91 3941443,
E-mail:quico@eucmax.sim.ucm.es
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)
Trang 2M A T E R I A L S A N D M E T H O D S
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) Pancreatic
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 mMHanks/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 incubated 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/Rcontain-inger bicarbonate, pH 7.2, enriched with
1% BSA and glucose (1.67, 5.5 or 16.7 mM), and placed in a
metabolic incubator in an atmosphere of 95% O2and 5%
CO2 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
radioimmuno-assay method of Herbert et al [15], using rat insulin as a
standard
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 acid/guanidinium isothiocyanate method
[16] Total RNA was size-fractionated through a 1.3%
agarose/formaldehyde gel and 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 pGEM7
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 pancreatic 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¢) were designed to amplify a 564-bp frag-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 each 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 for 3 s at 4 °C in 100 lL lysis buffer containing 5% SDS,
80 mMTris/HCl, pH 6.8, 5 mMEDTA, 10% glycerol and
1 mM phenylmethanesulfonyl ¯uoride The lysates were centrifuged, and the protein contents of the resulting supernatants were measured by the Bio-Rad protein assay kit Liver pieces were homogenized in buffer containing
1 mM Tris/HCl, pH 7.5, 1 mM MgCl2, 10 lM phenyl-methanesulfonyl ¯uoride, 1 lgálL)1 leupeptin and 3 mM 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 mMTris/HCl, pH 7.4, 150 mMNaCl) 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 rabbit IgG or an anti-sheep IgG conjugated 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
Trang 3Determination of mitochondrial enzyme activities
As a ®rst step to the determination of citrate synthase,
succinate dehydrogenase and cytochrome c oxidase
activ-ities, isolated pancreatic 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 10
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 lL 0.75M Tris/
HCl, pH 8.0, was mixed with 100 lL 1% Triton X-100,
100 lL 5,5¢-dithiobis(2-nitrobenzoic acid), 50 lL
acetyl-CoA (7 mgámL)1) and 10 lL tissue homogenate, and then
made up to 950 lL with distilled water and incubated at
30 °C for 2 min A412was measured for the last minute in a
Beckman DU-68 spectrophotometer Then, oxalacetate was
added to initiate the biochemical reaction, and rates of
absorbance were recorded for 2±3 min To calculate the
enzyme activity, we subtracted the change in A412before the
addition of oxalacetate (which is acetyl-CoA hydrolase
activity) from the change in A412after addition of
oxalace-tate
Succinate dehydrogenase activity [18] was determined
after mixing 40 lL tissue homogenate with 500 lL Krebs/
Ringer phosphate buffer, pH 7.0, containing 100 lL
dichloroindophenol and 50 lL KCN, and then made up
to 900 lL with water Then 100 lL 320 mM succinate
was added and the D600was measured for 5 min at 30 °C
Cytochrome c oxidase activity [18] was determined after
the incubation of 100 lL Krebs/Ringer phosphate buffer,
pH 7.0, 100 lL 1% reduced cytochrome c, and 780 lL of
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 D550was measured for
2 min
Mitochondrial enzyme activities (i.e citrate synthase,
succinate dehydrogenase, and cytochrome c oxidase) were
expressed as nmolámin)1á(mg protein))1 In our hands,
analytical variation coef®cients of mitochondrial enzyme
activities were below 10% and recoveries ranged from 90%
to 120% The protein contents of the samples were
determined by the method of Lowry et al [19]
R E S U L T S
Effect of glucose concentration on insulin release
by pancreatic islets of fetal, suckling and adult rats
Table 1 shows that an increase in the concentration of
glucose in the incubation medium from 5.5 to 16.7 mM
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 mM 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 ofGLUT-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 2A) 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 even higher than in suckling 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 Eect of dierent 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 pgá(ng DNA) )1 á2 h )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.
Trang 4Effect of glucose concentration on the expression
ofGLUT-2 and glucokinase mRNA and protein
in the pancreatic islets of fetal and adult rats
We studied the effect of glucose concentration (2.8, 5.5 and
20 mM) on GLUT-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 mM
In addition, when fetal islets were preincubated with 2.8 mM glucose and then incubated with 20 mM glucose, the expression of GLUT-2 mRNA increased signi®cantly In contrast, preincubation of fetal islets with 20 mM glucose which were then incubated with 2.8 mMglucose produced the opposite effect GLUT-2 mRNA levels in fetal pancre-atic islets cultured with 20 mMglucose 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 dierent
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 analyses of GLUT-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 dierent experimental groups Densitometric data were calculated as the percentage of adult values Values are means SEM from four independent experiments *P < 0.001 compared with adults.
Trang 5were 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 in the culture medium was increased When fetal islets were switched from 2.8 to 20 mM glucose, the expression of GLUT-2 increased up to 10-fold In contrast, when fetal islets were switched from 20 to 2.8 mMglucose,
an 85% reduction in protein content was observed Similarly, an increase in glucose concentration in the culture medium stimulated the expression of the glucokinase mRNA and protein of fetal pancreatic 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 of all three enzymes were always lower in 21-day-old fetuses and 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 experimental groups, 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 half that found 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 content of 10%
in fetal rats and 35% in 10-day-old suckling rats relative to the values found in adult animals
D I S C U S S I O N
During adult life, pancreatic b cells are able to recognize changes in circulating nutrients, mainly glucose, through a sensor system that facilitates the metabolism of glucose in these cells and then the synthesis and secretion of insulin In contrast with these events, fetal pancreatic b cells secrete insulin poorly in response to increased glucose concentra-tions in the extracellular space [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 the top of both panels are the bands corresponding to the
dierent 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.
Trang 6expressed much later in liver than in pancreatic 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 controlled by glucose [24],
whereas insulin appears to be the major positive effector of
glucokinase activity in liver [23,24] We also found that, in
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 Accordingly, 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 Eect 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 dierent 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 dierent glucose concentrations (2.8, 5.5 and 20 m M ) or 20 m 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 speci®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 of GLUT-2 in fetal pancreatic islets cultured with dierent 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 means 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 three panels are the bands corresponding to the dierent experimental groups Densitometric data were calculated as the percentage of adult rats Values are means SEM from four independent experiments *P < 0.05, **P < 0.005 compared with adults.
Trang 7We also observed that the highest amounts 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 other 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 mM) in 19, 20 and 21-day rat fetuses [4], respectively, compared with the 5.5 mM 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 to pregnant rats induces maturation of fetal pancreatic b cells so that they release insulin in response to glucose [32]
Because the rat is born in an immature state, we studied the activities of three mitochondrial enzymes to obtain information about the number 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 Eect 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 dierent 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 glucokinase
and 18S rRNA Densitometric data express the glucokinase/18S
rRNA ratio as the percentage of the values obtained at 5.5 m M
glucose (B) Western-blot analysis of glucokinase in fetal pancreatic
islets cultured with dierent glucose concentrations (2.8, 5.5 and
20 m M ) for 48 h Densitometric data were calculated as the
per-centage of the values obtained at 5.5 m M glucose Values are
mean-s SEM from three independent experimentmean-s *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-pared with adult values.
Trang 8activity, 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 is mGDH, de®ciencies in
the activity or contents 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 glucose concentrations may not be due to modi®ed functioning of ATP-dependent K+ and voltage-dependent Ca2+ channels because different insulin secretagogues, but not glucose, increase intracellular
Ca2+in fetal rat b cells [36] However, 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
A C K N O W L E D G E M E N T S
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 This investigation was supported by grants from the DireccioÂn General de InvestigacioÂn Cientõ®ca y TeÂcnica (DGI-CYT), the Fondo de InvestigacioÂn Sanitaria de la Seguridad Social, and the Comunidad de Madrid, Spain.
R E F E R E N C E S
1 Asplund, K., Westman, S & Hellerstrom, C (1969) Glucose stimulation of insulin secretion from the isolated pancreas of foetal and newborn rats Diabetologia 5, 260±262.
2 BlaÂzquez, E., Lipshaw, L.A., BlaÂzquez, M & FoaÁ, P.P (1975) The synthesis and release of insulin in foetal, nursing and young adult rats: studies in vivo and in vitro Pediatr Res 9, 17±25.
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Fig 8 Ontogenesis of mGDH protein in rat pancreatic islets and liver.
Western-blot analyses of mGDH in pancreatic islets (A) and liver (B)
from 21-day-old fetuses (F-21), 5, 10, and 20-day-old suckling (5,
S-10, and S-20) and adult rats At the top of the panels are the bands
corresponding to the dierent experimental groups Densitometric
data were calculated as the percentage of adult rats Values are
means SEM from four independent experiments *P < 0.05.
**P < 0.001 compared with adult values.
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