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Endocrinology Vol. 140, No. 8 3762-3766
Copyright © 1999 by The Endocrine Society

ARTICLES

Expression and Regulation of Glucokinase in Rat Islet ß- and {alpha}-Cells during Development1

Jian Tu, Bernard E. Tuch and Zhaoyi Si

Pancreas Transplant Unit, The Prince of Wales Hospital, Faculty of Medicine, The University of New South Wales, Sydney 2031, Australia

Address all correspondence and requests for reprints to: Jian Tu, M.D., Ph.D., Pancreas Transplant Unit, Department of Endocrinology, The Prince of Wales Hospital, High Street, Randwick, New South Wales 2031, Australia. E-mail: j.tu{at}unsw.edu.au


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Glucokinase (GK) is the rate-limiting enzyme in the glycolytic pathway of the ß-cell and, even in the rat fetus at 22-days gestation, immediately before birth, acts as a sensor of glucose influencing the rate of glucose utilization. However, when GK first appears in islets during ß-cell development is unknown. Whether GK is expressed in fetal glucagon-producing cells is also unknown. To determine this information, fetal rat islets were examined at 16-, 18-, and 22-days gestation. GK was identified immunocytochemically in both ß- and {alpha}-cells at all these ages, with the number of GK immunoreactive cells positively correlated to the fetal age from 16–22 days. Western blot analysis of islet protein extracts demonstrated the presence of GK, at 52 kDa, at 16 days and thereafter. To determine whether glucose had any effect on regulation of GK biosynthesis, fetal islets were cultured in medium containing a wide range of concentrations of glucose for 7 days. The amount of GK protein was significantly decreased in low concentrations of glucose and augmented at high concentrations. In conclusion, GK was expressed in both ß- and {alpha}-cells in fetal rat islets during development. GK is an integral part of the function of both of these cells at all stages in the development of the fetal islet.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN THE MATURE pancreatic ß-cell and hepatocyte, glucokinase (GK) plays a dominant role in the regulation of glucose homeostasis by catalyzing the rate-limiting biochemical reaction of glycolysis. It regulates glucose-induced insulin secretion from the ß-cell and is responsible for maintaining a gradient for glucose transport into hepatocytes (1, 2, 3, 4). This enzyme first appears in the rat hepatocyte, 16 days after birth (5, 6), at a time when the diet changes from milk [which is high in fat (7)] to solids [which are rich in carbohydrate (8)]. In contrast, GK is present in the rat islet at 20–21 days of fetal life (9), but exactly when it first appears in islets is unknown.

It was believed that GK was present in the mature pancreas only in ß-cells. Heimberg et al. (10) have recently shown that this enzyme is expressed also in mature glucagon-producing {alpha}-cells. This has been disputed by others (11, 12). Which cells in the fetal rat islet produce GK are unknown, although it is presumed that ß-cells do, because culturing islets in high concentrations of glucose up-regulates activity of the enzyme (9). To answer this question, we have examined fetal rat islets immunocytochemically at 16-, 18-, and 22-days gestation for GK, as well as insulin and glucagon. Furthermore, regulatory expression of GK protein by glucose in these developing islet cells was studied. These experiments shed light on the role of GK in fetal islet ß- and {alpha}-cells during development.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
All chemicals were of analytical grade and were obtained from Sigma Chemical Co. (St. Louis, MO). All enzymes were purchased from Roche Molecular Biochemicals (Mannheim, Germany). [5-3H]-D-glucose and 3H2O were acquired from NEN Life Science Products, Inc. (Boston, MA).

Islet preparation
Fetal pancreata were obtained from pregnant Wistar inbred rats at gestational ages 16, 18, and 22 days (crown-rump length 22 ± 1, 37 ± 1, and 45 ± 1 mm, respectively). Approval to conduct experiments was obtained from the Animal Care and Ethics Committee of the University of New South Wales. Pancreata from within a litter were pooled, minced, and subsequently digested in 0.8 mg/ml collagenase A. Islets, which appeared as golden translucent colored orbs, were handpicked, the number from each fetal pancreas being 40–100.

Adult rat islets were isolated from the pancreata of 10-week-old Wistar male rats by a modification of the collagenase method described previously (13). Briefly, the glands were distended by injection of 1 mg/ml collagenase P and then digested in two steps (14). Islets were separated from exocrine tissue by Ficoll gradient (9). For each experiment, 2400 islets (obtained from five rats) were used.

Islet culture
Islets were maintained, at 37 C in an atmosphere of 95% air-5% CO2, in nonattachable Petri dishes containing 25 ml of antibiotic free RPMI 1640 medium, 10% heat-inactivated FCS, and glucose at a concentration of 1.4, 2.8, 5.6, 11.2, or 16.8 mM. Culture medium was changed every second day, for a maximum of 7 days. Islets were removed at the end of culture, for determination of GK activity and glucose utilization and Western blot analysis.

Immunocytochemistry
GK-expressing cells were identified immunocytologically in fetal rat pancreata. Sixteen-, 18-, and 22-day-old fetal rat pancreata were fixed in 4% paraformaldehyde, for 12 h at 4 C, before being embedded in paraffin (11). Consecutive 5-µm sections were deparaffinized with 100% histolene and successively in 100, 95, 90, 85, and 75% ethanol. We used 3% hydrogen peroxide to block endogenous peroxidase and 5% skim milk to block nonspecific binding. GK-expressing cells were identified by staining with a polyclonal sheep antiserum against a purified B1 isoform of rat GK (a gift from Dr. M. A. Magnuson, Dept of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN), followed by biotinylated antibody, peroxidase-conjugated streptavidin (DAKO Corp. Laboratories Inc. Carpinteria, CA), and 3,3'-diaminobenzidine (Sigma Chemical Co.) as substrate. For dual staining, primary antibodies to insulin or glucagon (DAKO Corp.) were applied to the sections after GK staining. The secondary antibody was biotinylated, followed by alkaline phosphatase-conjugated streptavidin and new fuchsin (DAKO Corp.) as substrate. The coverslip was mounted in glycergel (DAKO Corp.). Primary antibody was omitted for negative controls, and adult rat pancreas and liver were used as positive controls. A minimum of 2000 islet cells with visible nuclei was examined in each section.

Western blot analysis
GK protein was identified by Western blot, as described previously (9). The only difference from the technique described previously was that protein extract from fetal rat islets was concentrated with Microcon-10 (Amicon Inc., Beverly, CA), an instrument used to separate molecules at a molecular mass cutoff of 10 kDa. Only molecules with molecular mass greater then this were used in the Western blot. Islet protein, in 9-µg aliquots, was loaded onto SDS-PAGE containing 10% acrylamide and was electrotransferred onto a nitrocellulose filter (Hoefer Scientific Instruments, San Francisco, CA) (9). A polyclonal sheep antiserum (1:1,000) against a rat GK was used as the primary antibody, followed by donkey antisheep IgG conjugated to alkaline phosphatase (1:5,000) (Silenus Laboratories, Hawthorn, Australia). Rat liver extracts, prepared in the same manner as pancreatic islets, were used as positive controls for GK. Intensity of the GK band was quantitated by computerized image analysis (Gel Doc 1000 Gel Documentation System, Bio-Rad Laboratories, Inc., Hercules, CA) using MultiAnalyst software (Bio-Rad Laboratories, Inc.).

Size of GK protein
The molecular weight of GK protein was confirmed by silver staining as described previously (9). A mixture of islet proteins and prestained SDS-PAGE standards was examined by this technique. Rat liver protein extract was stained as a positive control for GK. Molecular weight was determined by computerized image analysis using MultiAnalyst software.

Statistical analysis
Data are expressed as the mean ± SEM. Statistical differences among different groups were determined by the unpaired two-tailed Student’s t test and ANOVA if the variances were equal and by the Mann-Whitney nonparametric test if variances were unequal (15). P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
ß- and {alpha}-cells express GK at all fetal ages
GK was observed to be present in islets at all gestational ages examined (Fig. 1Go and Fig. 2Go, C and D), with the enzyme colocalized in cells expressing both insulin and glucagon (Fig. 2Go). Most of these cells, but not all of them, contained this enzyme (Fig. 2Go and Table 1Go), with intensity of expression being heterogeneous (Figs. 2Go, D and F).



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  		Figure 1. GK staining of fetal rat islets at 16- (A) and 18-days (B) gestational age. Consecutive 5-µm pancreatic sections were exposed to a GK antibody. GK-positive cells are stained brown. The scale bar represents 20 µm.

 


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  		Figure 2. Immunocytochemical staining of fetal rat islets at 22-days gestational age. Consecutive 5-µm pancreatic sections A, C, E and B, D, F were immunostained with an antibody to insulin (A) and glucagon (B) to localize islet ß- and {alpha}-cells, respectively, and to a GK antibody (C, D). Dual staining of insulin and GK, glucagon and GK are shown in E and F, respectively. Insulin- and glucagon-positive cells are stained red and GK-positive cells brown. The scale bar represents 20 µm.

 

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  		Table 1. Percentage of insulin-, glucagon-, and glucokinase-positive cells

 
The percentage of insulin- and GK-positive cells increased linearly with fetal age from 16 to 22 days (r = 0.99, P < 0.001). Further, there was a positive correlation between the number of insulin-positive and GK-positive cells in fetal islets (r = 0.99, P < 0.001). The percentage of glucagon-positive cells in islets remained constant during fetal life.

Expression of GK protein
Silver staining showed a major band at 52 kDa, the molecular mass of GK, in freshly isolated 16-, 18-, and 22-day-old fetal and adult islets. Immunoblotting confirmed that this band was caused by GK (Fig. 3Go).



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  		Figure 3. Western blot for GK in freshly isolated rat islets from (lane a) 16-day-old fetal islets, (b) 18-day-old fetal islets, (c) 22-day-old fetal islets, and (d) adult islets.

 
Regulation of GK by glucose
The ability of glucose to regulate the expression of GK protein, in fetal islets obtained at 22-days gestation, was examined by culturing the tissue in medium containing various glucose concentrations (1.4–16.8 mM) for 7 days. Results are shown in Fig. 4Go, A and B. The intensity of the GK band was positively related to the concentration of glucose (r = 0.99; P < 0.001).



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  		Figure 4. Glucose induction of GK protein. A, Western blot for GK on 9 µg of protein extract from 22-day-old fetal rat islets cultured at different concentrations of glucose for 7 days; B, intensity (%) of GK band is expressed as the mean ± SEM of four experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our results demonstrate expression of GK, in both ß- and {alpha}-cells in the fetal rat islets, from as early as 16-days gestation (Table 1Go), which is the earliest time when the islet has developed (16, 17). The presence of GK early in gestation is consistent with the major role glycolysis plays throughout fetal life, when anaerobic metabolism is preferred to oxidative metabolism (18, 19). Histological analysis of the graphs suggests that GK is present in greater amounts in {alpha}-than ß-cells (Fig. 2Go, D and F). These findings are similar to those obtained in purified adult rat {alpha}- and ß-cells by others (10), with higher levels of GK protein in {alpha}- than ß-cells, when GK was detected by Western blots.

In fetal rat islets, even as early as 16 days, the ß-cell is the dominant endocrine cell type, and we have found that it is this cell in which GK is mostly expressed (Table 1Go). Similarly, the ß-cell is the main cell type in adult rat islets, and it is this cell in which GK is also mostly expressed (12).

Our data show that the percentage of glucagon-containing {alpha}-cells in the fetal rat islets is 12%, and this figure is constant from 16- to 22-days gestation. These data are different from those of McEvoy (17), who demonstrated in 1980 that {alpha}-cells were the dominant cell type in 16-day-old fetal rat islets, with 96% of cells being of this type, with the number reduced to 48% at 18-days and 32% at 22-days gestation. A possible explanation for these differences between us and McEvoy is the specificity of the antibody being used in immunocytochemical staining. It is possible that antiglucagon serum being raised by McEvoy (17) recognized other islet cells.

A heterogeneous pattern of distribution of GK among not only ß-, but also {alpha}-cells in developing fetal rat islets (Figs. 1Go and 2Go), suggests functional variability of fetal ß- and {alpha}-cells. This conclusion would be supported if there were a positive relationship between the amount of GK and glucose utilization. Our biochemical data show this. Both the amount of GK protein (Fig. 4Go) and glucose utilization (9) are positively correlated to the glucose concentration of the culture medium. Hence, the level of GK protein and glucose utilization is positively correlated. Functional diversity among adult ß-cells, but not {alpha}-cells, has previously been noted, with variable expression of GK (12, 20) and the insulin promoter (21), sensitivity to glucose (21, 22, 23, 24), proinsulin synthesis (25), membrane potential (26), insulin content (27), and insulin secretion (28).

That the prevailing concentration of glucose was positively correlated to the concentration of GK protein is consistent with GK being the glucose sensor in the fetal rat ß-cell. Previously, we had reached the conclusion by showing a positive correlation between GK activity and glucose concentration, as well as between glucose utilization and glucose concentration (9). It is possible that GK is also a glucose sensor in the fetal {alpha}-cells. Thus, the up-regulation of GK we observed during culture in the presence of high concentrations of glucose may have occurred in both ß- and {alpha}-cells, leading to enhanced glucose utilization in the former cell and probably diminished glucagon secretion in the latter (10).

In summary, we have demonstrated that GK is expressed in fetal rat islet ß- and {alpha}-cells from 16-days gestation. GK is an integral part of the function of these cells at all stages in the fetal islet during development.


   Footnotes
 
1 This work was supported by a Project Grant from the National Health and Medical Research Council of Australia. Back

Received December 1, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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