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The Journal of Clinical Endocrinology & Metabolism Vol. 88, No. 6 2753-2759
Copyright © 2003 by The Endocrine Society

Insulin Secretagogues, But Not Glucose, Stimulate an Increase in [Ca2+]i in the Fetal Human and Porcine ß-Cell

Anthony J. Weinhaus, Muhammad T. Tabiin, Philip Poronnik, Catalina A. Palma, David I. Cook and Bernard E. Tuch

Departments of Medicine (A.J.W., B.E.T.) and Physiology (A.J.W., P.P., D.I.C.), University of Sydney, NSW 2006; and Diabetes Transplant Unit (M.T.T., C.A.P., B.E.T.), Prince of Wales Hospital, University of New South Wales, Sydney, NSW 2031, Australia

Address all correspondence and requests for reprints to: B. E. Tuch, M.D., Ph.D., Diabetes Transplant Unit, Prince of Wales Hospital, High Street, Randwick, NSW 2031 Australia. E-mail: b.tuch{at}unsw.edu.au.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Fetal pancreatic ß-cells release insulin poorly in response to glucose; however, the cellular mechanism for this is unknown. By using fura-2 to measure changes in the cytoplasmic free Ca2+ concentration in ß-cells, we examined human/porcine fetal islet-like cell clusters (ICCs) and human adult islets for the presence of functional K+ATP and voltage-activated Ca2+ ion channels. The effects of glucose, glyceraldehyde, leucine, KCl, and the channel effectors glipizide and BAY K8644 were studied. In fetal human/porcine ICCs and adult islets, KCl, glipizide, and BAY K8644 increased [Ca2+]i. Both glucose and glyceraldehyde increased [Ca2+]i in islets but had no effect on ICCs. Leucine increased [Ca2+]i in islets and porcine but not human ICCs. We hypothesize that the beneficial effect of leucine in fetal porcine, but not human ICCs, is attributable to time-dependent maturation of the ß-cells, because porcine ICCs examined were at 87% of the gestational period, and human ICCs were at 42%.

Our data demonstrate that both K+ATP and voltage-activated Ca2+ channels, required for glucose-stimulated increase in [Ca2+]i, are functional early in gestation. This suggests that the cause of the immaturity of fetal human/porcine ß-cells is at a more proximal step of glucose-induced metabolism than the channels on the cell surface.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IT HAS BEEN ESTABLISHED that insulin secretion from fetal human (1, 2) and porcine (3, 4) pancreatic ß-cells is poorly responsive to glucose, compared with adult cells. The exact reasons that underlie this immaturity remain unclear, despite the efforts of a number of researchers (5, 6, 7, 8).

In mature ß-cells, the cellular events that lead from an increase in extracellular glucose concentration to the secretion of insulin are well understood. After glucose transport into the cell, glucose is metabolized via glycolysis, the tricarboxylic acid cycle, and oxidative phosphorylation, resulting in an increase in the production of ATP. The increase in the cytosolic ATP/ADP ratio results in the blocking of the K+ATP channels in the plasma membrane (9), thereby inhibiting K+ efflux and depolarizing the membrane potential (10), which leads to the opening of the L-type Ca2+channels (11). Thus, ATP-induced depolarization of the membrane results in the influx of Ca2+ into the cell and the subsequent elevation of [Ca2+]i, the signal that triggers insulin release (1, 12, 13, 14, 15).

Agents that elevate [Ca2+]i in human fetal ß-cells, such as KCl and ionomycin, have been shown to promote insulin secretion (1), indicating that insulin secretory granules are able to migrate to the cell surface and discharge their content. Therefore, the immaturity of the fetal ß-cell must lie before this step.

It has been shown that the K+ATP channels and L-type Ca2+ are present and functional at 16 d of gestation in the fetal rat ß-cell (16). It can therefore be concluded that the inability of the rat fetal ß-cell at these gestational times (76% of gestation) to secrete insulin in response to glucose is not attributable to the absence or malfunctioning of the channels. Whether this is true earlier in gestation is unknown. Human fetal ß-cells are available at a much earlier gestational age than is the rat, 14–19 wk (35–48% gestation). In this manuscript, we have examined human fetal ß-cells, using the radiometric fluorescent probe of Ca2+ (fura-2): 1) to determine whether the cells respond with an increase in [Ca2+]i to glucose and other secretagogues that stimulate insulin secretion in mature cells; and 2) to characterize the pathways involved.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals

L-arginine HCl, D- and L-leucine, D-glyceraldehyde, dibutyryl cAMP, and the disodium salt of ATP were purchased from Sigma (St. Louis, MO). Pure glipizide was kindly provided by Farmitalia Carlo Erba Pty Ltd. (Milano, Italy). BAY K8644 (±enantiomeric) was obtained from Calbiochem (San Diego, CA). All of the chemicals were of the highest available grade.

Pancreatic tissue

Human fetal pancreases were obtained from the therapeutic termination of pregnancies carried out by suction curettage between 14- and 20-wk gestation. Maternal consent and ethical approval from the Institutional Ethics Committees were obtained for the use of the tissue.

Human adult pancreases were obtained from a nondiabetic 10-yr-old (undergoing distal pancreatectomy for removal of an insulinoma) and nondiabetic adult cadaver donors. Porcine fetal pancreases were obtained from the uteri removed from pregnant sows after slaughter and removed aseptically from each fetus. The gestational age of the fetuses ranged from 80–115 d (term is 118 d).

Isolation of pancreatic islet-like cell clusters (ICCs)

Human fetal ICCs were isolated, following the procedure described previously (17). Briefly, pancreases were finely minced and digested in 4.0–6.0 mg/ml type P collagenase (Roche Molecular Biochemicals, Mannheim, Germany) in Hank’s balanced salt solution, at 37 C for 7 min, in a shaking water bath. The digested tissue was then washed twice in ice-cold Hank’s balanced salt solution, then twice in culture medium, before being centrifuged for 4 min at 800 rpm. The culture medium consisted of RPMI-1640 plus 10% fetal calf serum, supplemented with 10% amino acids solution (X50), plus the addition of gentamicin and penicillin at 50 µg/ml. The supernatant was then removed, and the pellet was resuspended in culture medium, plated onto Petri dishes, and cultured for 3 d at 37 C in 5% CO2 in air. The ICCs were then handpicked with a micropipette and cultured for an additional 2–5 d at 37 C in 5% CO2 in air, on nonattachable Petri dishes, in the culture medium previously described. After culture, the ICCs were prepared for microfluorometric studies.

Isolated islets from adult human pancreases were prepared using the same techniques as those for isolation of fetal ICCs, but the collagenase concentration was 2 mg/ml, the digestion time was 30 min (18), and the total time that the islets were in tissue culture was 1 d.

Isolated ICCs from fetal pig pancreases were prepared using the same technique as that for human fetal ICC isolation, but the collagenase concentration was 4 mg/ml, the digestion time was 20 min, and the concentration of fetal calf serum in the culture medium was 7% (3). After handpicking of the ICCs on d 3, the ICCs were cultured for an additional 4–6 d in the same manner as the human fetal ICCs.

Loading and perfusion of pancreatic ICCs

Isolated ICCs were loaded for 60 min at 37 C with 15 µg/ml of the fluorescent intracellular dye fura-2 AM (Molecular Probes, Inc., Eugene, OR) plus 10 µg/ml 20% (wt/vol) stock Pluronic F-127 solution (Molecular Probes, Inc.) in a shaking water bath. After incubation, the ICCs were washed three times. The solution used for loading, washing, and perfusion consisted of the following (in mM): NaCl, 145; KCl, 5; MgCl2, 1; H-HEPES, 20; CaCl2, 2; D-glucose, 2 (pH adjusted to 7.4 by NaOH at room temperature) (16).

Single, whole ICCs were transferred individually to a closed perfusion chamber of approximately 300 µl vol and were perfused at a flow rate of 1 ml/min by a peristaltic pump (Minipuls, Gilson Inc.). All experiments were performed at room temperature.

Measurements of [Ca2+]i

Microfluometric experiments were performed using a Nikon Diaphot inverted microscope (Nikon, Tokyo, Japan) equipped with x40 Fluor objective that allowed the entire ICC to be contained within the field of view. Fura-2-loaded ICCs were irradiated alternately with light at 340 and 380 nm, using a filter wheel. The light emitted from the loaded cells was passed through a 505 ± 10-nm bandpass filter, detected by a photomultiplier; and the resultant signal was recorded on a 4-channel MacLab (ADInstrument Pty Ltd., Sydney, Australia). Fura-2 ratios (R) were calculated as 340/380 nm and were converted into [Ca2+]i, according to the equation derived by Grynkiewicz et al. (19). The dissociation constant for fura-2 at room temperature was taken to be 135 nM (19). For calibration, the cells were exposed to a 150-mM KCl solution plus 2 µM ionomycin (pH 8.0) (20), containing either 10 mM EGTA (Rmin) or 10 mM CaCl2 (Rmax).

Insulin secretion and content

Ability of fetal ß-cells to secrete insulin in response to glucose was examined by static stimulation of ICCs in air, as described previously (21). Briefly, human and porcine ICCs, in multiples of 200, were washed with PBS (1 mM CaCl2, 0.5 mM MgCl2, 26.7 mM NaHCO3, 20 mM HEPES) containing 0.2% BSA (Sigma) and exposed in quadruplicate to this basal (PBS + 2.8 mM glucose) or stimulated (PBS + stimulus) buffer, for 1 h at 37 C. The stimuli used were: 20 mM glucose, 20 mM KCl, 50 µM glipizide, 2 µM BAY K8644, 20 mM leucine, 1 mM cAMP, 20 mM glyceraldehyde, 20 mM arginine, and 100 µM ATP. At the end of the hour, cells were pelleted by centrifugation, and the supernatant was analyzed for insulin content. Acid ethanol was added to the pellet to extract intracellular insulin, and this suspension was also assayed for insulin. Insulin levels were determined by RIA, using a human insulin standard, kindly provided by Novo Nordisk A/S (Bagsvaerd, Denmark).

Statistical analysis

Results are expressed as mean ± SEM (number of observations). Statistical significance was assessed by using the Student’s unpaired t test, or ANOVA with data normalized by log transformation. Duncan’s test was then used to separate the groups (P < 0.05).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
[Ca2+]i measurements

Fetal human ICCs. Fetal human ICCs had a resting [Ca2+]i of 46 ± 5 nM (n = 30, from 10 different pancreases). Exposure of fetal ICCs to a 10-min pulse of 20 mM glucose failed to elicit a change in [Ca2+]i (Fig. 1AGo, Table 1Go). Similarly, exposure of the fetal ICCs to 20 mM glyceraldehyde (Fig. 1BGo, Table 1Go) or to 20 mM leucine (Fig. 1BGo, Table 1Go) failed to cause a significant increase in [Ca2+]i above resting levels. In adult ß-cells, glyceraldehyde and leucine (like glucose) are metabolized to form ATP, thereby increasing the ATP/ADP ratio and closing the K+ATP channels, resulting in an increase in [Ca2+]i (22). These results suggest that there is a deficiency in the metabolism and/or ATP synthesis; and therefore, glucose, glyceraldehyde, and leucine were unable to cause a blockage of K+ATP channels, membrane depolarization, or subsequent activation of the L-type Ca2+ channels.



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  		Figure 1. The effects of the nutrients glucose (A), glyceraldehyde and leucine (B), and the sulfonylurea glipizide (A and B) on [Ca2+]i in single human fetal ICC. The addition of glyceraldehyde (20 mM), leucine (20 mM), and glipizide (50 µM) to the perifusion solution and the increase in glucose concentration (from 2 to 20 mM) are indicated by the black bars. The expected straight baseline has been included in dots (A).

 

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  		Table 1. Summary of the stimulus-induced increases in [Ca2+]i from human fetal, human adult, and porcine fetal ICC

 
We used channel effectors to directly confirm the presence of K+ATP and L-type Ca2+ channels. In fetal ICCs, glipizide (50 µM), a K+ATP channel blocker in adult ß-cells (23), caused a rapid increase in [Ca2+]i of 60 ± 11 nM above resting levels (Fig. 1Go, A and B; Table 1Go), presumably by depolarization-induced Ca2+ influx. This is strong evidence for the presence of K+ATP channels in these cells.

Direct evidence of the presence of L-type Ca2+ channels in fetal ß-cells was provided by a known L-type Ca2+ channel agonist, BAY K8644 (10, 24). Exposure of fetal ICCs to 2 µM BAY K8644 caused a rapid increase in [Ca2+]i of 57 ± 8 nM (Fig. 2AGo, Table 1Go). This is strong evidence for the presence of L-type Ca2+ channels in fetal ICCs.



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  		Figure 2. The effects of arginine and BAY K8644 (A) and extracellular ATP exposure (B) on [Ca2+]i in human fetal ICCs. The addition of arginine (20 mM), BAY K8644 (2 µM), and ATP (100 µM) to the perifusion solution is indicated by the black bars.

 
Additional evidence, albeit indirect, for the presence of the L-type Ca2+ channels was demonstrated by exposing the fetal ICCs to arginine and KCl. The cationic amino acid, arginine, is transported electrogenically into the ß-cell (25), causing depolarization and a subsequent increase in [Ca2+]i (11, 26). Exposure of fetal ICCs to 20 mM arginine caused a rapid, transient increase in [Ca2+]i of 37 ± 7 nM (Fig. 2AGo, Table 1Go), thus demonstrating that membrane depolarization results in an increase in [Ca2+]i. Increasing the extracellular concentration of K+ ions in ß-cells leads to the depolarization of the plasma membrane potential and activation of the L-type Ca2+ channels (11). Exposure of fetal ICCs to 20 mM KCl caused a rapid, transient increase in [Ca2+]i of 111 ± 36 nM (Table 1Go), providing additional evidence that membrane depolarization results in the activation of the L-type Ca2+ channels and an increase in [Ca2+]i.

To provide evidence that the human fetal ICC is responsive to glucose-independent mechanisms, the cells were exposed to a stimulator of intracellular Ca2+ release. It has been well described in rat (27, 28) and human (29) ß-cells that the exposure to extracellular ATP stimulates the production of inositol-triphosphate in the cytosol, which then causes the release of Ca2+ from intracellular stores, resulting in an increase in [Ca2+]i. Exposure of fetal ICCs to extracellular ATP (100 µM) caused a rapid, transient increase in [Ca2+]i of 304 ± 47 nM above resting levels (Fig. 2BGo, Table 1Go).

Human adult islets. The resting [Ca2+]i in the adult islet was 76 ± 13 nM (n = 11, from three different pancreases), values comparable with those reported by others (10). In contrast to the lack of response shown by fetal ICCs to glucose, adult islets responded to 20 mM glucose, with an increase in [Ca2+]i of 50 ± 28 nM (Fig. 3AGo, Table 1Go). This increase in [Ca2+]i had a lag time of 239 ± 51 sec after the introduction of glucose. As reported previously (29), the increase in [Ca2+]i in response to 20 mM glucose was preceded by an initial, transient decrease (not shown).



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  		Figure 3. The effects of the nutrients glucose (A), glyceraldehyde (B), and leucine (C) and the channel effectors BAY K8644 (A and C) and glipizide (B) on [Ca2+]i in single human adult islets. The addition of glyceraldehyde (20 mM), leucine (20 mM), BAY K8644 (2 µM), and glipizide (50 µM) to the perifusion solution and the increase in glucose concentration (from 2 to 20 mM) are indicated by the black bars.

 
Adult islets also responded to 20 mM glyceraldehyde (Fig. 3BGo), whose [Ca2+]i increase had a lag time of 117 ± 29 sec. As expected, there was a response to a number of other stimuli; these were: 20 mM KCl, 50 µM glipizide (Fig. 3BGo), 2 µM BAY K8644 (Fig. 3CGo), and 20 mM leucine (Fig. 3CGo). The [Ca2+]i response to these stimuli is shown in Table 1Go.

Porcine fetal ICCs. The above data from human fetal ICCs, obtained very early in gestation (35–50% of term), demonstrate that the end-stage of the signal transduction pathway in the human fetal ß-cell is mature. The lack of a response to glucose, glyceraldehyde, and leucine indicates that the pathways of glucose oxidation at a later stage in metabolism are incomplete or not functional. To determine whether maturation occurred during gestation, studies are needed on ß-cells obtained late in gestation. Human fetuses are not available at this time for research purposes; but tissue from the fetal pig, late in gestation, is. Pigs are physiologically very similar to humans, with pig insulin being 97% homologous with human insulin. Accordingly, fetal porcine pancreatic tissue obtained at 76–97% of term was used.

Fetal porcine ICCs had a resting [Ca2+]i of 37 ± 4 nM (n = 16, from seven different pancreases). Exposure of fetal ICCs to a 10-min pulse of 20 mM glucose failed to elicit a change in [Ca2+]i (Fig. 4AGo, Table 1Go). In contrast to the lack of response to glucose, exposure of the fetal ICC to 20 mM leucine caused an increase in [Ca2+]i above resting levels of 85 ± 42 nM (Fig. 4AGo, Table 1Go), the increase occurring 50 ± 12 sec after the introduction of leucine. Unlike the human fetal ICC, these results demonstrate that the porcine fetal ICC responds to leucine, with an increase in [Ca2+]i as a result of ATP synthesis, leading to the blockage of K+ATP channels, membrane depolarization, and subsequent activation of the L-type Ca2+ channels.



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  		Figure 4. The effects of the nutrients glucose (A), glyceraldehyde (B), and leucine (A) and the channel effectors glipizide (B), KCl (C), and BAY K8644 (C) on [Ca2+]i in single fetal porcine ICC. The addition of glyceraldehyde (20 mM), leucine (20 mM), glipizide (50 µM), KCl (20 mM), and BAY K8644 (2 µM) to the perifusion solution and the increase in glucose concentration (from 2 to 20 mM) are indicated by the black bars. The expected straight baseline has been included in dots (B).

 
Exposure of the porcine fetal ICCs to glipizide (50 µM) caused a rapid increase in [Ca2+]i of 58 ± 15 nM (Fig. 4BGo, Table 1Go). Likewise, exposure of the fetal ICCs to BAY K8644 (2 µM) caused an increase in [Ca2+]i of 78 ± 15 nM (Fig. 4CGo, Table 1Go). These data provide strong direct evidence for the presence of both the K+ATP channels and the L-type Ca2+ channels in the fetal porcine ß-cells.

Additional evidence for the presence of the L-type Ca2+ channels was demonstrated by the response of the fetal porcine ICCs to an increased extracellular KCl concentration. Exposure of fetal ICCs to 20 mM KCl caused a rapid, transient increase in [Ca2+]i of 68 ± 20 nM (Fig. 4CGo, Table 1Go).

Insulin secretion. All agents that caused an increase in [Ca2+]i also enhanced insulin secretion. (Table 1Go). Furthermore, agents that had no effect on [Ca2+]i did not affect the level of insulin secretion.

ß-Cells from human and porcine fetal ICCs secreted insulin in response to KCl, glipizide, BAY K8644, and cAMP (Fig. 5Go, A and B) but not to glucose or glyceraldehyde. Arginine and ATP were tested on human fetal ICCs and found to enhance insulin secretion. Leucine enhanced insulin secretion from porcine (but not human) fetal ICCs.



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  		Figure 5. Insulin secretion of human (A) and porcine (B) fetal ICCs in response to glucose (Gluc, 20 mM), KCl (20 mM), glipizide (Glip, 50 µM), BAY K8644 (BAY K, 2 µM), leucine (Leu, 20 mM), cAMP (1 mM), glyceraldehyde (Glycer, 20 mM), arginine (Arg, 20 mM), and ATP (100 µM). *, P < 0.05. Data was collected from nine pancreases. For each agent tested, n = 18 (A), and n = 21 (B).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have demonstrated, by monitoring changes in [Ca2+]i with fura-2, that fetal human ICCs possess the complete mechanism for translating an increase in the cytosolic ATP/ADP ratio to an increase in [Ca2+]i. The increase in [Ca2+]i has been described to be an essential requirement for insulin release from the human fetal ß-cell (1). The difference that we observed between fetal ICCs and adult islets was that glucose and leucine failed to elicit an increase in [Ca2+]i in the fetal cells. This result is in agreement with the previously reported finding that the human fetal ICCs release insulin poorly in response to glucose (1, 2) and leucine (1).

This report demonstrates the presence of K+ATP and L-type Ca2+ channels in human fetal ICCs by their responsiveness to insulin secretagogues. Glipizide was found to increase [Ca2+]i in fetal ICCs by acting on K+ATP channels, whereas KCl and BAY K8644 were found to increase [Ca2+]i by acting on L-type Ca2+ channels. In a similar manner, we also show that fetal porcine ICCs have functional K+ATP and L-type Ca2+ channels. These data extend information obtained from previous studies of fetal rat islets, obtained later in gestation (76% of gestation), which demonstrated, by microfluometric analysis (16) and patch-clamping experiments (7), the presence of such channels. Our results also agree with those of other groups studying adult ß-cells, who have shown (using electrophysiological and fluorescent techniques) that glucose, leucine, and other secretagogues cause an increase in [Ca2+]i in rat (7, 11, 12, 22) and human islets (10, 29, 30, 31).

Though the majority of Ca2+ channels in ß-cells are thought to be of the L-type (32), there are also T-type, or transient, channels. Unlike the L-type Ca2+ channels, the T-type channels are not involved in insulin secretion (33). Transient spikes in [Ca2+]i, which are characteristic of T-type Ca2+ channels, were not seen in the experiments conducted, suggesting the absence of T-type channels.

Fetal pancreatic ICCs contain a heterogeneous cell population with 8% ß-cells and 4% {alpha}-cells (see Ref. 35) scattered among a large number of protodifferentiated duct cells (34, 35). The increase observed in [Ca2+]i in response to the sulfonylurea glipizide demonstrates that measurements were taken from ß-cells, and not other pancreatic cell types, because glipizide acts exclusively in the pancreas on the ß-cells. Furthermore, results of insulin responsiveness to stimuli (36) parallel the [Ca2+]i responses for each of the stimuli. This supports the view that changes in fura 2 were being recorded mostly from ß-cells, because insulin is secreted solely from these cells. Some changes in fura 2, especially those attributable to ATP, may have occurred in {alpha}-cells, which also posses K+ATP and L-type Ca2+ channels.

Our results, showing that the human fetal ICCs do not respond to glucose, glyceraldehyde, or leucine, indicate that insufficient ATP is synthesized, after exposure to these nutrients, to initiate the series of events required to cause insulin secretion. Because glucose transport and glycolysis are normal in human fetal ß-cells (5, 37), the level of immaturity must be distal to glycolysis. This could be in the conversion of lactate to pyruvate, in the TCA cycle, and/or in the shuttles that transport H+ from the cytoplasm to the mitochondria (8).

Porcine fetal ICCs are responsive to stimulation by leucine, an amino acid that enters the TCA cycle, resulting in production of sufficient ATP to initiate insulin secretion. However, the inability of glyceraldehyde to stimulate a change in the [Ca2+]i suggests that the pathways between glyceraldehyde-3-phosphate and mitochondrial oxidation, and/or in the glycerol phosphate dehydrogenase shuttle, are not fully expressed or functional. Very recent studies on fetal porcine ß-cells confirm that this hydrogen ion shuttle is immature (8).

Fetal porcine ß-cells have a level of maturity greater than that of the human fetal ß-cells. The difference between the fetal cells of human and pig can be explained by considering the relative gestational periods of each species. As stated previously, porcine fetal tissue was obtained at 76–97% gestation, and human fetal at 35–48% gestation. An alternative hypothesis to this time-dependent maturation is a difference in effect between human and pig. Species differences are unlikely, because fetal rat islets of 76% gestation were examined and found to be responsive to leucine (16). To be absolutely certain, examination of this porcine fetal tissue early in gestation (35–48%) will be necessary.

We can speculate, therefore, from observations made with ß-cells throughout gestation, that the development of the glucose-stimulated ß-cell response occurs in phases from mid to late gestation. At all stages, the K+ATP and L-type Ca2+ channels are functional. By 80% of gestation, mitochondrial oxidation is functional, but there is a lack of coupling factors to glucose utilization (8). By birth, these factors are functional. Full responsiveness to glucose thereafter (38) may require higher expression of glucose transporter 2, which is low in fetal and suckling rat ß-cells (6, 39).

In conclusion, human fetal ß-cells, acquired during the second trimester, at 35–48% of gestation, possess functional ATP-dependent K+ and L-type Ca2+ channels. These cells are able to secrete insulin in response to stimulation that activates these channels. It may be concluded, therefore, that the lack of glucose-induced insulin secretion from fetal ß-cells early in gestation does not seem to be attributable to the absence or malfunctioning of the channels.


   Acknowledgments
 
The expert technical assistance of S. Beynon, A. Simpson, J. Madrid, and J. Tu in tissue preparation is gratefully acknowledged.


   Footnotes
 
This work was supported by a project grant from the National Health and Medical Research Council of Australia; an Overseas Postgraduate Research Award from the Australian Department of Employment, Education and Training (to A.J.W.); and a University of Sydney Postgraduate Research Award (to A.J.W.).

Abbreviations: ICC, Islet-like cell cluster; R, fura-2 ratios.

Received October 2, 2002.

Accepted February 21, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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