Expression of glucose transporter-2, glucokinase and mitochondrial glycerolphosphate dehydrogenase in pancreatic islets during rat ontogenesis Marta Garcõ  a-Flores 1 , Jose  Antonio Zueco 1 , Joaquõ  n Arenas 2 and Enrique Bla  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  zquez, Departamento de Bioquõ  mica, 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) 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  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  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  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. 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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. 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,