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Expression of Glucokinase in Glucose-Unresponsive Human Fetal Pancreatic Islet-Like Cell Clusters¹

Department of Endocrinology, The Prince of Wales Hospital, Sydney, Australia

Address all correspondence and requests for reprints to: Bernard E. Tuch, M.D., Ph.D., Department of Endocrinology, The Prince of Wales Hospital, High Street, Randwick, New South Wales 2031, Australia.

	Abstract

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Glucokinase (GK) is the glucose sensor in the adult ß-cell, resulting in fuel for insulin synthesis and secretion. Defects in this enzyme in the ß-cell are responsible for the genetic disorder maturity-onset diabetes of the young, with the ß-cell being unable to secrete insulin appropriately when challenged with glucose. The human fetal ß-cell is also unable to secrete insulin when exposed to glucose, but whether GK is present and functional in this developing cell is unknown. To determine the expression of GK in human fetal pancreatic tissue, cytosolic protein was extracted from human fetal islet-like cell clusters (ICCs) at 17–19 weeks gestation and examined for protein content and enzyme activity. On Western blots, a single band corresponding to GK was seen at 52 kDa, and this was similar to that obtained from human adult islets. The maximal velocity (V_max) of GK was less in fetal ICCs than that in adult islets (8.7 vs. 20.7 nmol/mg protein·h); similar K_m values were found in both ICCs and islets. No attempt was made to determine which cells in an ICC contained GK. Glucose utilization was determined radiometrically; the V_max of the high K_m component was less in ICCs than in islets (31.3 pmol/ICC·h vs. 101.4 pmol/islet·h). Culture of ICCs for 3–7 days in medium containing 11.2 mmol/L glucose resulted in a 3.7-fold increase in the V_max of GK and a 1.8-fold increase in glucose utilization. These enhanced activities of glucose phosphorylation and glycolysis, however, did not lead to the ß-cell being able to secrete insulin when exposed to glucose. In conclusion, glucokinase is present and functional in human fetal ICCs, but the inability of the human fetal ß-cell to secrete insulin in response to an acute glucose challenge is not due to immaturity of this enzyme.

	Introduction

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IN MATURE ß-cells, glucose causes insulin secretion. Glucose is passively transported into the pancreatic ß-cells, where it is phosphorylated by glucokinase (GK). The metabolism of glucose-6-phosphate by glycolysis and tricarboxylic acid cycle results in ATP production. It is ATP that closes ATP-dependent K⁺ channels on the cell surface, causing depolarization of the cell membrane, opening of the L-type Ca²⁺ channels, and influx of Ca²⁺, which leads to exocytosis of insulin granules. GK, the first key enzyme in the glycolytic pathway, controls the phosphorylation of glucose to glucose-6-phosphate, and is the glucose sensor in the adult ß-cell (1, 2). The regulatory effect of glucose on GK is at the translational level (3). This enzyme is also the glucose sensor in the rat fetal islet late in gestation (4). Whether the same is true in the human fetal islet-like cell cluster (ICC) or proislet, which is formed by collagenase digestion of fetal pancreas obtained during the early part of the second trimester, is not known. For that matter it has not been established that GK is actually present or functional in the fetal pancreas at this early stage of gestation, although very low levels of messenger ribonucleic acid for GK have been detected at 13 weeks gestation (5).

Maturity-onset diabetes of the young is a subtype of NIDDM with an early age of onset, autosomal dominant inheritance, and impaired secretion of insulin in response to glucose. This disorder is due to defects in the gene for GK, resulting in reduced activity of GK (6). The human fetal ß-cell is also unable to secrete insulin when challenged with glucose (7, 8, 9), although it is able to synthesize and store insulin (7, 8, 9) and secrete it in response to agents that elevate levels of cAMP (8, 10, 11), increase intracellular levels of Ca²⁺, and activate the enzyme protein kinase C (8, 10, 11, 12). The following study was undertaken to investigate whether GK protein was expressed in human fetal ICCs and, if so, to determine its activity in glucose phosphorylation and its contribution to glycolysis.

	Materials and Methods

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Materials

All chemicals were of analytical grade and were purchased from Sigma Chemical Co. (St. Louis, MO). All enzymes were obtained from Boehringer Mannheim (Mannheim, Germany); D-[5-³H]glucose and ³H₂O were acquired from DuPont-New England Nuclear Research Products (Boston, MA).

Preparation of ICCs

Human fetal pancreas was obtained from the therapeutic termination of pregnancies between 16–19 weeks gestation (40–48% of term), and ICCs were isolated as described previously (11). Maternal consent for the use of fetal tissue was obtained, and ethical permission was given by the human ethics committees of both The University of New South Wales and the Eastern Sydney Area Health Service (Sydney, Australia).

Preparation of islets

Human adult islets were prepared from the pancreas obtained from nondiabetic adult cadaveric organ donors and islets isolated as described previously (13).

Immunocytochemistry

The percentage of insulin-positive cells in ICCs was determined immunocytochemically using laser-scanning confocal microscopy (14). Two hundred ICCs were first fixed in 4% paraformaldehyde and then placed on gelatin-coated glass microscope slides before being fixed in ice-chilled 70% ethanol overnight at 4 C. The cells were permeablized with 0.1% Triton and 1% fetal calf serum (FCS), then blocked with 0.1% Triton and 3% FCS. Insulin-containing cells in ICCs were identified by staining with guinea pig antiinsulin primary antibody (Dako Laboratories, Carpenteria, CA), followed by rhodamine-conjugated antiguinea pig secondary antibody (Sigma). The coverslip was mounted in glycerol-phosphate-buffered saline (19:1). Primary antibody was omitted, and sections were incubated with 1% guinea pig serum for negative controls. Rhodamine-labeled sections were excited at 515 nm and viewed with a 530-nm barrier filter (Olympus Laser Scanning Confocal Microscope, Olympus Optical Co., Tokyo, Japan). ICCs were scanned every 0.5–0.7 µm from top to bottom, and the percentage of insulin-positive cells was calculated.

The percentage of ß-cells in islets was determined immunocytochemically using a light microscope. Islets were fixed in 1.5% glutaraldehyde (pH 7.2) for 1 h at 4 C and embedded in paraffin. Sections of this sample were placed on gelatin-coated glass microscope slides and allowed to attach before being fixed in ice-chilled 100% methanol. The slides were washed with phosphate-buffered saline, covered with hydrogen peroxide for 10 min, and stained with guinea pig antiinsulin antibody, followed by rabbit antiguinea pig antibody, biotinylated antirabbit antibody, and then streptavidin (Dako Laboratories). The chromogen used was 3-amino-9-ethylcarbazole (Sigma), and the counterstain was hematoxylin. Primary antibody was omitted for negative controls, and adult rat pancreas sections were stained as positive controls. A minimum of 1000 cells with visible nuclei was examined in each section.

Western blot analysis

GK protein was identified by Western blot as described previously (4). The only difference from the technique described previously was that protein extracts from fetal ICCs or adult islets were concentrated with Microcon-10 (Amicon, Beverly, MA), an instrument used to separate molecules at a molecular mass cut-off of 10 kDa. Only molecules with molecular masses greater than this were used in the Western blot; their protein contents were twice that of the original extract. Twenty micrograms of these concentrated protein components were resolved by SDS-PAGE containing 10% acrylamide and electrotransferred onto a nitrocellulose filter (Hoefer Scientific Instruments, San Francisco, CA) (4). A polyclonal sheep antiserum (1:1000) against a purified B1 isoform of rat GK (gift from Dr. M. A. Magnuson, Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN) was used as the primary antibody, followed by donkey antisheep IgG conjugated to alkaline phosphatase (1:5000; Silenus Laboratories, Hawthorn, Australia). Protein extract from 4-week-old pig hepatocytes prepared in the same manner as ICCs/islets was used as a positive control for GK, and prestained SDS-PAGE standards (Bio-Rad, Richmond, CA) were included as molecular mass controls.

Size of GK protein

The molecular mass of GK protein was confirmed by silver staining, as described previously (4). Concentrated protein extracts of fetal ICCs or adult islets and prestained SDS-PAGE standards were examined by this technique. Protein extract from 4-week-old pig hepatocytes was stained as a positive control for GK.

Glucose phosphorylation

Glucose phosphorylation was quantitated by measuring the rate of glucose-6-phosphate formation in a modification of the fluorometric assay with glucose-6-phosphate dehydrogenase as described previously (4, 15). One thousand randomly chosen freshly isolated or cultured fetal ICCs or adult islets were examined by this technique after they were homogenized on ice in 80 µL homogenization buffer (pH 7.7) containing 20 mmol/L K₂HPO₄, 1 mmol/L ethylenediamine tetraacetate, 110 mmol/L KCl, and 5 mmol/L dithiothreitol.

Glucose utilization

Glucose utilization was determined by measuring the formation of ³H₂O from D-[5-³H]glucose as described previously (4, 16). For this analysis, 100 randomly chosen freshly isolated or cultured fetal ICCs or adult islets were used for each concentration of glucose.

Insulin secretion

Fetal ICCs or adult islets were centrifuged through a Percoll layer of density 1.04 g/mL to remove debris and dead cells. Batches of 100 fetal ICCs or 40 adult islets were taken, and insulin secretion from them was determined as described previously (4), but a human insulin standard (Novo Research Institute, Bagsvaerd, Denmark) was used in the RIA (17).

Statistical analysis

Data are expressed as the mean ± SEM (number of experiments). Statistical differences between groups were determined by the unpaired two-tailed Student’s t test and ANOVA if variances were equal or the by Mann-Whitney nonparametric test if variances were unequal (18). P < 0.05 was considered significant.

	Results

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Percentage of ß-cells in ICCs and islets

Immunocytochemical analysis of human fetal ICCs (Fig. 1) showed that 6.4 ± 1.1% of cells (200 ICCs from 3 preparations) contained insulin, a figure similar to that reported previously by other groups (19, 20, 21). In contrast, 47 ± 2% of human adult islets (1000 from eight preparations) contained insulin, a figure also similar to that reported previously by other groups (2, 22).

View larger version (109K): [in this window] [in a new window]   Figure 1. Confocal image of an insulin-stained human fetal ICC. The ICC was scanned every 0.5–0.7 µm from the top of the ICC to the bottom. Bar = 50 µm.

Western blot and silver staining showed a major single band at 52 kDa, which corresponds to the GK protein, in the concentrated protein extract from human fetal ICCs and adult islets; an identical band was seen in 4-week-old pig hepatocytes (Fig. 2). The intensity of GK in the human fetal ICCs was 88.1 ± 5.2% of the band area compared to 100 ± 4.3% in the same gel for adult islets (n = 4–6).

View larger version (41K): [in this window] [in a new window]   Figure 2. Western blot for GK. Lane a, Ten micrograms of cytosolic protein extract from 4-week pig hepatocytes; lanes b–d, 20 µg concentrated protein from 18-week human fetal ICC extract (b), a 34-yr-old male human donor islet extract (c), and an 18-yr-old male human donor islet extract (d).

The two components of glucose phosphorylation activity are depicted in Fig. 3. They are a high K_m activity characteristic of GK and a low K_m representing hexokinase (HK) (23). The kinetic parameters of GK and HK in the human fetal ICCs and adult islets are detailed in Table 1. The V_max of GK was significantly less in freshly isolated human fetal ICCs (P < 0.05), but when allowance was made for the fewer number of insulin-containing cells in ICCs than islets, the V_max was similar in the two groups. The V_max of HK and the K_m values of both GK and HK were similar in fetal and adult tissue.

View larger version (15K): [in this window] [in a new window]   Figure 3. Kinetic analysis of glucose phosphorylation in fetal ICCs (A) and adult islets (B). Glucose phosphorylation activity was assayed by a fluorometric method, and data were plotted according to the method of Eadie-Hofstee. A representative of three separate experiments in shown. GK (•) and HK () activities are displayed together in the same graph. The inset is a representation of data for the high Km component (GK) using a magnified scale of the relevant part of the x-axis.

Glucose utilization

The results of glucose utilization and its kinetics are summarized in Table 2 and Fig. 4. In the high K_m system, the V_max of glucose utilization in the fresh fetal ICCs was significantly less than that in adult islets (P < 0.001), but when allowance was made for the smaller number of insulin-containing cells in ICCs, their V_max was significantly greater (P < 0.05). The V_max of ICCs increased 1.8-fold when the clusters were exposed to medium containing 11.2 mmol/L glucose for 3–7 days. The K_m values in both fetal groups were significantly less than those of fresh adult islets (P < 0.05).

View larger version (14K): [in this window] [in a new window]   Figure 4. Glucose utilization in freshly prepared and cultured fetal ICCs as well as adult islets incubated in Krebs-Ringer bicarbonate buffer containing a wide range of glucose concentrations for 1 h. A representation of three separate experiments is shown: fresh () and cultured (•) fetal and adult (). The inset represents data for the low Km system using a magnified scale of glucose concentration from 0.05–0.3 mmol/L glucose.

Insulin secretion

Insulin secreted from freshly isolated fetal ICCs and adult islets stimulated by 0, 1.7, and 16.7 mmol/L glucose and by 16.7 mmol/L glucose plus 1 mmol/L 8-bromo-cAMP is depicted in Fig. 5. At 0 mmol/L glucose, adult islets secreted 0.5 ± 0.01% of their cellular insulin content hourly. This remained the same (0.6 ± 0.01%) in the presence of 1.7 mmol/L glucose, was 5 times higher (2.9 ± 0.01%) in 16.7 mmol/L glucose, and was 11 times greater (6.2 ± 0.02%) when 1 mmol/L 8-bromo-cAMP was added to the higher concentration of glucose. Fetal ICCs did not respond to 16.7 mmol/L glucose, but did respond to a combination of 1 mmol/L 8-bromo-cAMP and 16.7 mmol/L glucose, with insulin secretion being double the basal rate. The insulin content of fetal ICCs was less than that of adult islets (275 ± 4 µU/ICC vs. 1204 ± 367 µU/islet; n = 4; P < 0.001).

View larger version (18K): [in this window] [in a new window]   Figure 5. Insulin secretion from human fetal ICCs and adult islets expressed both as raw results (A) and as a percentage of insulin content (B). Data represent the mean ± SEM of four to six experiments (, fetal; , adult). a, P < 0.02 compared with 0 and 1.7 mmol/L glucose; b, P < 0.002 compared with 0, 1.7, and 16.7 mmol/L glucose; c, P < 0.03 compared with 16.7 mmol/L glucose.

	Discussion

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This study is the first report to demonstrate the expression of GK protein in glucose-unresponsive human fetal ICCs. It supplements a previous report showing GK messenger ribonucleic acid in human fetal pancreas of 13 weeks gestational age (5). GK protein was identified at a molecular mass of 52 kDa, which is identical to that found in human adult islets and pig hepatocytes. The amount of GK protein in fetal ICCs was similar to that obtained from human adult islets, suggesting that both ß- and non-ß-cells in ICCs contained GK, as the number of ß-cells in ICCs was 6.4% compared with 47% in adult islets. The presence of GK in non-ß-cells of the pancreas has been reported previously in adult rat -cells (24). That the amount of GK protein was similar in ICCs and islets, yet GK activity was lower in ICCs, is possibly due to nonfunctional GK protein expressed in fetal undifferentiated cells, which constitute the majority of cells in an ICC, -cells or other endocrine cells. It is theoretically possible that nonfunctional GK is also present in some ß-cells. The lower V_max of GK in the freshly isolated ICCs was probably due to a reduced number of ß-cells in ICCs, as when the number of ß-cells in ICCs was corrected to that in adult islets, the difference was no longer observed. This and the comparable affinities of GK in ICCs and islets suggest that GK is functioning in an adult manner in fetal pancreatic ß-cells.

In the high K_m component of glucose utilization, which indicates activity of the anaerobic glycolytic pathway, V_max was lower in ICCs than adult islets; when allowance was made for the smaller number of insulin-containing cells in ICCs, the V_max was greater in ICCs. This latter finding is similar to our recent report for the late gestational fetal rat islets, in which the V_max for the high K_m of glucose utilization was greater in fetal than adult islets (4). These results indicate that anaerobic glycolysis is favored in fetal life, a finding reported previously for nonpancreatic tissues (25, 26). The V_max that we obtained in adult islets (101.4 ± 8.0 pmol/islet·h) was different from that reported by Ashcroft et al. (27) (85.0 ± 15.6 pmol/islet·90 min, or 56.6 ± 10.4 pmol/islet·h). This difference can be explained by the maximal concentration of glucose in which islets were incubated, 16.7 mmol/L glucose used by Ashcroft (27) and 30 mmol/L glucose used in our experiments. The rate of glucose utilization at 16.7 mmol/L glucose in our experiments was the same as that reported by Ashcroft. That the K_m of glucose utilization in adult islets was greater than that in fetal ICCs was not unexpected, as similar differences between adult and fetal islets have been reported previously in rats (4).

Fetal ß-cells secrete little or no insulin when exposed to glucose (28). One explanation advanced to explain this is a reduction in the activity of GK (29, 30, 31). Our data from uncultured ICCs do not support this hypothesis. Further evidence against any connection between GK and immaturity of fetal ß-cells comes from experiments culturing ICCs for 3–7 days in medium containing 11.2 mmol/L glucose. The resultant 3.7-fold increase in the V_max of GK and the 1.8-fold increase in glucose utilization did not result in any glucose-induced insulin secretion. A possible explanation for the immaturity of fetal ß-cells is insufficient glucose oxidative phosphorylation in mitochondria with inadequate synthesis of ATP and, hence, failure to close ATP-dependent K⁺ channels and initiate the cascade of events in signal transduction required for the secretion of insulin (30, 32).

In summary, we have demonstrated the presence of 52-kDa GK in human fetal ICCs in the second trimester of pregnancy (40–47.5% of term), an age much earlier than that reported previously in late gestational fetal rat islets (95% of term) (4). That the amount of GK protein was similar in ICCs and islets suggests that non-ß-cells also contained GK, since the number of ß-cells in the former was only one seventh of that in the latter. The lesser activity of this enzyme in ICCs suggests that GK was functional in only a percentage of cells containing this enzyme, probably the ß-cells. Up-regulation of GK activity by culturing fetal ICCs in 11.2 mmol/L glucose for 3–7 days did not result in glucose responsiveness in insulin secretion, indicating that the inability of the human fetal ß-cell to secrete insulin in response to glucose is not due to a deficiency of GK.

	Acknowledgments

 
The authors thank Ms. S. Beyon for preparing human fetal ICCs, Mr. P. Waugh for carrying out confocal microscopy of human fetal ICCs, and Dr. J. Sun of the Liver Transplantation Unit of Royal Prince Alfred Hospital (Sydney, Australia) for providing adult pig hepatocytes.

	Footnotes

 
¹ This work was supported by the National Health and Medical Research Council of Australia.

² Recipient of an Overseas Postgraduate Research Award from the Australian Department of Employment, Education, Training, and Youth Affairs.

Received September 18, 1996.

Revised November 8, 1996.

Accepted November 25, 1996.

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This Article Abstract Full Text (PDF) Submit a related Letter to the Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Tu, J. Articles by Tuch, B. E. Search for Related Content PubMed PubMed Citation Articles by Tu, J. Articles by Tuch, B. E.