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Am J Physiol Endocrinol Metab 292: E1018-E1029, 2007. First published December 5, 2006; doi:10.1152/ajpendo.00457.2006
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Physiological development of insulin secretion, calcium channels, and GLUT2 expression of pancreatic rat beta-cells

Victor Navarro-Tableros, Tatiana Fiordelisio, Arturo Hernández-Cruz, and Marcia Hiriart

Department of Biophysics, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Mexico City, Mexico

Submitted 29 August 2006 ; accepted in final form 16 November 2006


   ABSTRACT
TOP
 ABSTRACT
RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Insulin secretion in mature beta-cells increases vigorously when extracellular glucose concentration rises. Glucose-stimulated insulin secretion depends on Ca2+ influx through voltage-gated Ca2+ channels. During fetal development, this structured response is not well established, and it is after birth that beta-cells acquire glucose sensitivity and a robust secretion. We compared some elements of glucose-induced insulin secretion coupling in beta-cells obtained from neonatal and adult rats and found that neonatal cells are functionally immature compared with adult cells. We observed that neonatal cells secrete less insulin and cannot sense changes in extracellular glucose concentrations. This could be partially explained because in neonates Ca2+ current density and synthesis of mRNA {alpha}1 subunit Ca2+ channel are lower than in adult cells. Interestingly, immunostaining for {alpha}1B, {alpha}1C, and {alpha}1D subunits in neonatal cells is similar in cytoplasm and plasma membrane, whereas it occurs predominantly in the plasma membrane in adult cells. We also observed that GLUT2 expression in adult beta-cells is mostly located in the membrane, whereas in neonatal cells glucose transporters are predominantly in the cytoplasm. This could explain, in part, the insensitivity to extracellular glucose in neonatal beta-cells. Understanding neonatal beta-cell physiology and maturation contributes toward a better comprehension of type 2 diabetes physiopathology, where alterations in beta-cells include diminished L-type Ca2+ channels and GLUT2 expression that results in an insufficient insulin secretion.

insulin messenger RNA; calcium currents; functional immaturity

INSULIN SECRETION by pancreatic rat adult beta-cells is dependent on extracellular glucose concentration, whereas fetal beta-cells do not discriminate between different glucose levels. In fact, beta-cells of fetal mammals are considered immature, and animals depend on their mothers to regulate glucose homeostasis (20). It has been suggested that in neonatal islet cells the reduced insulin release in response to glucose is associated with a diminished increase in cytosolic Ca2+ concentration ([Ca2+]i) (6).

At birth, animals need to control glucose homeostasis in response to meals, and this could be a driving force for beta-cells to mature. Accordingly, it has been reported that, during the first days of life, rat beta-cells acquire most of the adult characteristics (16). However, the mechanisms and factors underlying the maturation process are still unclear.

Glucose-induced insulin secretion in adults is a Ca2+-dependent process, where membrane depolarization induced by glucose activates Ca2+ entry through different types of voltage-gated Ca2+ channels (VGCC) (25). Classically, L-type Ca2+ channels are considered the most important Ca2+ entry pathway controlling insulin secretion in rodent beta-cells, as well as in other species and beta-cell lines (8, 25, 32, 43). These Ca2+ channels open in response to strong depolarizations, conduct Ba2+ better than Ca2+, and do not inactivate during long-lasting voltage pulses (15).

T-type Ca2+ channels are present in beta-cells of rats and humans (15, 25, 29, 52), and they could be important for regulating insulin secretion as well as for triggering beta-cell death in response to cytokines (47).

Ca2+ channels are heteromeric protein complexes formed by {alpha}1-, beta-, {alpha}2-, {delta}-, and {gamma}-subunits. The {alpha}1 subunits form the ion-conducting pore, and Ca2+ channels are classified according to the type of {alpha}1 subunit expressed. beta-Cells from different species express different {alpha}1 subtypes; for example, adult rat beta-cells express {alpha}1 subunits responsible for generating the T, L, P/Q, and R types of VGCC (25). The {alpha}1 subunit that underlies the T-type Ca2+ current recorded in these cells corresponds to the {alpha}1G subunit (Cav3.1), which has been isolated and cloned in beta-cells (39, 41, 52).

On the other hand, the ion-conducting subunit that underlies the L-type Ca2+ current corresponds to {alpha}1D (Cav1.3) and {alpha}1C subunits (Cav1.2), which have been demonstrated in different types of beta-cells and beta-cell lines (1, 17, 23, 39). Most authors agree that the largest contributor of Ca2+ channels in rat beta-cells and related cell lines is the {alpha}1D subunit (3, 5, 27, 34, 40, 42, 43, 51). However, other authors describe the expression of the {alpha}1C subunit as being more important, especially in the mouse (18, 39, 40, 44).

The knockout mice {alpha}1D–/– develop postnatal hypoinsulinemia and glucose intolerance due to impaired expansion of the beta-cell mass (28). Those authors postulate that in the absence of {alpha}1D subunits knockout mice synthesize {alpha}1C subunits. Another study in INS-1 cells, where either {alpha}1D or {alpha}1C subunits were exclusively expressed, shows that the {alpha}1D subunit is preferentially linked to glucose-stimulated insulin secretion rather than the {alpha}1C subunit (23). In contrast, other groups suggest that {alpha}1C subunits are required for fast insulin secretion, associated with the first phase of insulin secretion in mice and RINm5F cells (1, 38, 40, 51).

To better understand postnatal beta-cell maturation, in this study we compared barium currents in isolated neonatal and adult beta-cells. We also analyzed glucose sensitivity, insulin synthesis and secretion, Ca2+ channel expression, and synthesis and molecular expression of {alpha}1 subunits forming VGCCs.


   RESEARCH DESIGN AND METHODS
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Reagents. Reagents were obtained from the following sources: collagenase type IV from Worthington (Freehold, NJ); bovine serum albumin (BSA), HEPES, poly-L-lysine, trypsin, and all salts of electrophysiological recordings from Sigma (St. Louis, MO); tissue culture dishes from Corning (Corning, NY); fetal bovine serum from Equitech-BIO (Ingram, TX); Hanks' balanced salt solution (HBSS), RPMI 1640 salts, and penicillin-streptomycin-amphotericin B solution from Life Technologies (Grand Island, NY); and tetrodotoxin from Calbiochem (La Jolla, CA).

Culture of pancreatic beta-cells. All methods used in this study were approved by the Animal Care Committee of the Instituto de Fisiología Celular, Universidad Nacional Autónoma de México. Animal care was performed according to the Guide for the Care and Use of Laboratory Animals (National Academy of Sciences, Washington, DC, 1996). Wistar rats were obtained from the local animal facility, maintained in a 14:10-h light-dark cycle (0600–2000), and allowed free access to standard laboratory rat diet and tap water. On the day of the experiments, animals were anesthetized with intraperitoneal pentobarbital sodium (40 mg/kg), and, after pancreas dissection, animals were killed by cervical dislocation.

Pancreatic beta-cells were obtained from neonatal (1-day-old) and young adult male rats (250–280 g), as previously described (28). Dissociation of the cells was achieved by incubating islets in a shaker bath for 10 min at 37°C in Ca2+-free Spinner solution, with 15.6 mmol/l glucose, 0.5% BSA, and 0.01% trypsin followed by mechanical disruption. Cells were cultured in RPMI 1640 (11.6 mmol/l glucose) supplemented with 1% fetal calf serum, 200 U/ml penicillin G, 200 µg/ml streptomycin, and 0.5 µg/ml amphotericin B. Before experiments, all islet cells were left in culture for 6–12 h, allowing them to recover from dissociation.

Frozen sections of pancreatic tissue. Pancreatic tissue was obtained from Wistar rats and fixed with 4% paraformaldehyde (PFH) in PBS for 12 h at 4°C. After a wash in PBS, pancreases were postfixed in buffered 4% PFH with 10, 20, and 30% sucrose (each for 24 h) and finally cut into 10-µm-thick sections with a cryostat (Leica CM 1900).

Insulin secretion. beta-Cells were cultured in control conditions for 24 h and then incubated for 30 min in HBSS (Sigma) supplemented with 5.6 mM glucose. Finally, cells were incubated for 1 h in HBSS with 5.6 or 15.6 mM glucose, and the medium was obtained and kept at –20°C until used. The amount of insulin secreted in such conditions was determined with a Mercodia Ultrasensitive rat insulin ELISA system (cat. no. 10-1137-10, ALPCO). Experiments were performed as instructed by the technical bulletin, using the supernatants of 5 x 105 beta-cells.

Double immunostaining of insulin and {alpha}1 subunits of VGCC or GLUT2. Cultures were fixed with 4% PFH in PBS for 45 min at room temperature, washed, and placed in a blocking solution containing 2% BSA (wt/vol) and 0.1% Triton X (vol/vol) for 30 min at room temperature. For pancreatic frozen sections, 1% (vol/vol) Triton was used instead. Samples were incubated in a humid chamber overnight at 4°C with primary antibodies raised against {alpha}1 subunits {alpha}1B, {alpha}1C, {alpha}1D, and {alpha}1G (anti-VGCC rabbit polyclonal IgG; Alomone Labs, Jerusalem, Israel), at 1:100 dilution for cultured cells and 1:10 dilution for frozen sections, and mouse anti-GLUT2 (Sigma) at 1:1,200 dilution for cells and 1:600 for frozen sections. The next day, samples were washed and incubated for 2 h at room temperature with the secondary antibody (1:50 dilution for frozen tissue and 1:100 for cultured cells), namely the cyanine 5 (Cy5)-conjugated F(ab')2 fragment of goat anti-rabbit IgG (Jackson ImmunoResearch). After a final washing with PBS, samples were incubated for 2 h with the primary guinea pig antibody anti-rat insulin (1:10,000 and 1:5,000 dilution for cells and frozen sections, respectively) and finally incubated for 1 h with the secondary antibody FITC-conjugated anti-guinea pig (1:100 dilution). Samples were mounted with medium containing 15 mM NaN3 (DAKO).

The following negative controls were done. 1) The primary antibodies (anti-VGCC or anti-insulin) were preadsorbed with the matching antigen (1 µg peptide/1 µg antibody). 2) Cell cultures were treated either with the primary antibody alone or 3) with the secondary antibody alone. As expected, these samples showed, in the first case, only background Cy5 or FITC fluorescence and no fluorescence in the other cases. Negative controls of the secondary antibodies were accomplished by incubating samples first with the primary anti-VGCC antibody, then with the Cy5-conjugated F(ab')2 fraction of goat anti-rabbit IgG (1:50 dilution), and finally with the unmatched secondary antibody (i.e., anti-guinea pig FITC). As expected, this procedure gave good Cy5 signal but no FITC signal.

Fluorescence imaging of cultured pancreatic cells and single-cell fluorescence quantification. For imaging of specific immunoreactivity, cultured cells were viewed under epifluorescence microscopy using a Nikon Diaphot inverted microscope equipped with a 100-W mercury lamp and a filter set appropriate for Cy5 (excitation 647 nm/DF32, emission 680 nm/DF32; Omega Optical, Brattleboro, VT) and FITC (excitation 488 nm/DF32, emission 522 nm/DF32; Omega Optical), and fluorescence images of each field were obtained separately. Samples were examined with high magnification using an oil immersion fluorescence objective (x63, 1.3 NA; Leitz Wetzlar, Germany). Digital images were acquired with a cooled CCD digital camera (SenSys 0401E; Roper Scientific, Tucson, AZ), and illumination was limited with a Uniblitz electronic shutter (Vincent Associates, Rochester NY). Exposures were chosen for the range of fluorescence intensities of each sample. Images were acquired with Image-Pro Express 2.0 software (Media Cybernetics, Silver Spring, MD) and stored in TIFF image format (8 or 12 bits resolution).

Image analysis was conducted with Image J 1.36 (Wayne Rasband; National Institutes of Health, Bethesda, MD) and MetaMorph 6.1 (Universal Imaging, West Chester PA).

For each pancreatic cell in the field, the cytoplasmic (nonnuclear) area was defined, and the mean fluorescence intensity was measured from this area of interest as well as from three to eight cell-free areas of the same coverslip (background fluorescence). Fluorescence measurements were performed automatically by the computer program over the selected areas of interest. The program measures the fluorescence signal from each pixel enclosed by the area of interest and computes the mean fluorescence over the total number of pixels. This automatically cancels any possible differences due to cell size. Background-corrected fluorescence intensities (specific fluorescence) were measured in all pancreatic cells identified in 200 fields from two independent cultures.

Data from these two cultures (20 animals per culture per each experimental condition) were pooled.

An assessment of the expression of Ca2+ channel protein in the plasma membrane relative to the cytoplasm was done as described by Viard et al. (46), with some modifications. Briefly, a line of 50 pixels was drawn on the image of each cell in the field, avoiding the cell nucleus (MetaMorph), and the intensity profile (fluorescence intensity measured over distance) was produced (see GoGoGoGoGoGoGoFig. 8). Similar profiles were drawn in ~25 cells from each condition. From each intensity profile, the mean fluorescence of five contiguous pixels centered at both edges of the cell were measured (plasma membrane signals) as well as from five contiguous pixels located in the middle of the cytoplasmic region (cytoplasmic signal). The membrane signals corresponding to both edges were averaged and divided by the mean cytoplasmic signal to give the membrane-to-cytoplasm (M/C) ratio. The mean M/C ratio from all cells was taken as representative of the staining pattern for a given condition. Numerical results were plotted with Origin 3.8 (Microcal, Northampton, MA), and image composition was made with Image J and Microsoft PowerPoint.


Figure 1
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  		Fig. 1. Insulin mRNA content and quantification of insulin content in neonatal (N) and adult (A) islet beta-cells in culture. A: representative images from Northern blot showing RT-PCR products and comparison of average levels of insulin mRNA expression in neonates (open bars) and adults (hatched bars). GAPDH, expression of glyceraldehyde-3-phosphate dehydrogenase. Data are expressed as means of 3 independent experiments (percentage vs. adult cells) ± SE. *P < 0.001 with respect to adults. B: representative fluorescence micrographs of neonatal and adult beta-cells in culture, showing differences in level of immunostaining. Scale bars, 15 µm. C: distribution histograms representing level of insulin-specific immunofluorescence measured from 538 neonatal and 490 adult cells. Distribution median is denoted by x.

 

Figure 2
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  		Fig. 2. Insulin secretion in response to glucose of neonatal and adult beta-cells. Determinations of insulin secretion from cultured neonatal (open bars) and adult beta-cells (hatched bars) in response to 5.6 and 15.6 mM glucose. Data are expressed as means ± SE of 3 independent experiments. *P < 0.001 with respect to adults cells; **P < 0.001 with respective control in 5.6 mM glucose.

 

Figure 3
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  		Fig. 3. Distribution pattern of GLUT2 transporters in neonatal and adult pancreatic islets in neonatal (A) and adult tissues (B). From left to right: insulin immunofluorescence (red), GLUT2 immunofluorescence (green), and merger of both images. Scale bar, 50 µm. C: pattern of GLUT2 immunofluorescence in cultured neonatal and adult beta-cells. Scale bar, 15 µm.

 

Figure 4
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  		Fig. 4. Comparison of Ba2+ currents in neonatal and adult pancreatic beta-cells. A: representative Ba2+ current families recorded from neonatal and adult beta-cells. B: Ba2+ current density (pA/pF) in freshly dissociated neonatal beta-cells (n = 12) is 30% smaller than in adult beta-cells (n = 10). C: comparison of voltage-dependence of activation of Ba2+ currents (see RESEARCH DESIGN AND METHODS). No significant differences were observed. Data are expressed as averages ± SE.

 

Figure 5
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  		Fig. 5. Expression of mRNAs coding for voltage-gated Ca2+ channels (VGCC) in islet beta-cells. Quantification was made by optical densitometry of the mRNA level in neonatal (open bars) and adult (hatched bars) beta-cells. mRNA expression levels were normalized against GAPDH mRNA levels. Representative images from Northern blot showing RT-PCR products as well as of GAPDH in neonatal and adult islet beta-cells are shown in the top of their respective {alpha}1B, {alpha}1C, {alpha}1D, and {alpha}1G subunit bars. Data are expressed as averages ± SE of 3 independent experiments. *P < 0.001 with respect to level of {alpha}1B subunit in neonatal cells; **P < 0.001 with respect to adults.

 

Figure 6
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  		Fig. 6. Double immunofluorescence for insulin and {alpha}1C or {alpha}1D subunits of VGCC in pancreatic islets. Representative confocal micrographs of pancreatic frozen sections showing islets of neonatal and adult rats. From left to right: insulin-specific staining (red), {alpha}1 subunit-specific staining (green), and merger of both images. Scale bars, 50 µm.

 

Figure 7
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  		Fig. 7. Double imunofluorescence for insulin and {alpha}1B and {alpha}1G subunits of VGCC in pancreatic islets. Representative confocal micrographs of pancreatic frozen sections showing islets of neonatal and adult rats. From left to right: insulin-specific staining (red), {alpha}1 subunit-specific staining (green), and merger of both images. Scale bars, 50 µm.

 

Figure 8
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  		Fig. 8. Subcellular distribution of immunoreactivity of {alpha}1D subunits of VGCC in beta-cells. Representative micrographs of cultured beta-cells from neonate (A) and adult (B). As these micrographs illustrate, the subcellular distribution differs between neonatal and adult beta-cells, with more intense immunostaining in the plasma membrane of mature cells. C and D: examples of fluorescence profiles obtained from white lines shown in A and B, respectively. See detailed explanation in RESEARCH DESIGN AND METHODS. Scale bars, 15 µm; line on the cell, 50 pixels (30 pixels = 10 µm).

 
Confocal imaging of frozen sections from pancreatic tissue. One day after immunostaining, all tissue slices from a given experiment were imaged with a laser scanning confocal microscope (MRC 1024; Bio-Rad, Hercules, CA) attached to a Nikon inverted microscope (Diaphot 300) equipped with a x40 oil immersion fluorescence objective (Nikon). Excitation and emission settings were as follows: for FITC staining, excitation 488 nm/DF32, emission 522 nm/DF32; for Cy5, excitation 647 nm/DF32, emission 680 nm/DF32. Imaging parameters remained fixed throughout the session. Images obtained at 488 and 647 nm excitation were collected either separately or concurrently. Subsequent analysis and processing of confocal images were carried out with software written by Todd Clark Breile, University of Minnesota (Confocal Assistant 4.02). For each experimental condition, ~10 islets from four tissue samples were analyzed.

Isolation of cytoplasmic RNA and RT-PCR detection of mRNA transcripts of Ca2+ channel subunits and insulin. Total RNA was extracted from fresh cells using TRIzol reagent (cat. no. 15596-026, GIBCO-BRL), as described in the manufacturer's technical bulletin (PerkinElmer cat. no. N808-0143). Cells were lysed directly in a culture dish by adding 1 ml of TRIzol reagent per 1 x 106 cells.

RT-PCR was carried out with 200 ng of total RNA for mRNA detection of either {alpha}1 subunits of VGCC or insulin. A parallel reaction was carried out in the same mRNA sample by using glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a housekeeping gene for quantitative purposes.

All oligonucleotide primers were synthesized and used to prime the amplification of the cDNA template. The sequences of the primers used by amplification were as follows: {alpha}1B 5'-AAAGCACAGAGCTTCTACTG-3' sense and 5'-GTGGTTGGA-GTCTCATCTTG-3' antisense; {alpha}1C 5'-AATCTCTTCTTGGCCATTGC-3' sense and 5'-CTGGAGGTCATCCATGTTGA-3' antisense; {alpha}1D 5'-GCTGAAAGTCTGA-ACACTGC-3' sense and 5'-AGCAGGAACCTCAGGCTCGT-3' antisense; {alpha}1G 5'-GAAGATGCGAGTGGACAG-3' sense and 5'-CTGTGGCGATGGTCACTG-3' antisense; insulin 5'-AAGAGCCATCAGCAAGC-3' sense and 5'-GAGCAGA-TGCTGGTGCAGC-3' antisense; GAPDH 5'-GCCCCCC-ATGTTTGTGAT-3' sense and 5'-GCCCCAGCATCAAAGGT-3' antisense. Thirty-five cycles of amplification were performed with an annealing temperature of 60°C and 35 cycles for {alpha}1B, {alpha}1C, and {alpha}1D; 67°C for {alpha}1G and 56°C for insulin and GAPDH with 16 and 20 cycles, respectively.

Reaction products were sequenced and proved to have 100% identity with the sequences reported for {alpha}1B, {alpha}1C, {alpha}1D, and {alpha}1G subunit, insulin, and GAPDH genes (data not shown). Amplified material was visualized by ethidium bromide staining following 1.5% agarose gel electrophoresis. Resolved PCR bands were analyzed by optical densitometry.

Electrophysiological recordings. The whole cell configuration of the patch-clamp technique (14) was used to record macroscopic voltage-gated Ca2+ currents, using Ba2+ as the charge carrier. Experiments were done at 20–22°C. The Axopatch 200B amplifier (Axon Instruments, Foster City, CA) was used, as previously described (7). Briefly, patch electrodes with a tip resistance of 1.5–3 M{Omega} were pulled from capillary tubes KIMAX-51 (Kimble Glass, Vineland NJ). Electrode tips were coated with Sylgard (Dow Corning, Midland, MI). The external solution consisted of (in mmol/l) 125 NaCl, 5 KCl, 2 MgCl2, 10 BaCl2, 10 HEPES, and 10 glucose. The internal solution contained (in mmol/l) 120 CsAsp, 10 CsCl, 5 CsF, 2.5 Cs-BAPTA, 10 HEPES, and 10 tetramethylammonium-Cl and 2.5 ATP. Na+ current was blocked by the addition of 100 nmol/l tetrodotoxin to the external solution.

The capacity transient of the pipette was canceled before the cell membrane was ruptured, and total cell capacitance was determined by digital integration of capacitive transients with +10-mV pulses from a holding potential of –80 mV. Cell capacitive transients were canceled, and series resistance was compensated by using the internal voltage-clamp circuitry. Remaining linear capacity transients as well as leakage currents were subtracted by a P/2 online procedure.

The pulse protocol used for the analysis of the Ba2+ currents (IBa2+) consisted of depolarizing test pulses of 15-ms duration, from –60 to +50 in 10-mV increments, from a holding potential of –80 mV. IBa2+ current activation curves were obtained by converting the peak current values to conductances.

Formula
where IBa2+ is the peak current value, Vm is the command pulse potential, and ErBa2+ is the apparent reversal potential, obtained by extrapolation of the I-V relationships, which in most cases was ~ +56 mV.

Conductance values were normalized and fitted to the Boltzmann relation:

Formula
where G is the IBa2+ peak conductance, Gmax is the maximal IBa2+ conductance, V1/2 is the midpoint of the activation curve, and k is the activation steepness factor.

Statistical analysis. All data are reported as means ± SE, where n denotes the number of cells or experiments. The statistical significance was obtained with the one-way analysis of variance (ANOVA) and Bonferroni analysis. The distribution of the fluorescence intensity in cell cultures follows a Poisson distribution rather than a normal distribution; therefore, a nonparametric statistical test (Kolmogorov-Smirnov) was used to compare data groups (10). Significant differences obtained with the Kolmogorov-Smirnov test are reported at the 95% confidence interval. A parametric statistic test (ANOVA factorial) was also used to compare data groups; significant differences obtained with this test are reported at the 95% confidence interval (Statview 4.57; Abacus Concepts, Cary, NC).


   RESULTS
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Insulin biosynthesis, content, and secretion in neonatal and adult beta-cells. Insulin mRNA was examined by RT-PCR. Neonatal beta-cells expressed nearly 40% less insulin mRNA than adult (Fig. 1A). However, when insulin immunofluorescence was examined, neonatal cells were clearly more immunoreactive than their adult counterparts. Examples are shown in Fig. 1B.

A rough assessment of insulin contained in individual beta-cells was possible by quantifying insulin-specific immunoreactivity in digital images (see RESEARCH DESIGN AND METHODS). Image analysis is shown in Fig. 1C, revealing two cell populations: cells with moderate immunoreactivity [between 6 and 50 arbitrary units (AU) of specific fluorescence], and cells with high immunoreactivity (i.e., >50 AU of specific fluorescence). The percentages of cells in these two groups were 90.6 and 9.4% in neonatal cells and 99.2 and 0.8% in adult cells, respectively. These data indicate that, on average, insulin immunoreactivity is stronger in the population of neonatal beta-cells compared with adult (medians are 26.9 and 13.6 AU, respectively). Assuming a linear relationship between immunolabeling and insulin content, this suggests that neonatal beta-cells contain nearly twofold more insulin than adult beta-cells.

Insulin secretion was examined by ELISA (see RESEARCH DESIGN AND METHODS) in neonatal and adult beta-cells by incubating cells for 1 h in both basal (5.6 mM) and stimulatory glucose concentration (15.6 mM). As illustrated in Fig. 2, insulin secretion from neonatal beta-cells at basal glucose was only 8% compared with that of adult cells, and it did not increase at the stimulatory concentration of glucose. In contrast, adult beta-cells secreted 79% more insulin when glucose increased from 5.6 to 15.6 mM.

These results suggest that neonatal beta-cells synthesize less insulin than adult cells. Nonetheless, the more than 10-fold difference in insulin secretion between adult and neonatal cells cannot be accounted for solely by insulin mRNA content. It is more likely that the key difference could reside in an inadequate coupling between glucose elevation and insulin secretion. In fact, this could explain why insulin tends to accumulate in the cytoplasm of immature beta-cells.

Expression of GLUT2 in neonatal and adult beta-cells. In addition to secreting less insulin, neonatal beta-cells fail to respond appropriately to the rise in extracellular glucose concentration. This could be due to a number of factors. A good possibility is the lack of sufficient glucose transporters (GLUT2), as has been previously observed (11, 13). As shown in Fig. 3, A and B, GLUT2 specific immunostaining is expressed in pancreatic frozen sections obtained from both neonatal and adult beta-cells. However, most of the immunoreactivity of the transporter appears to be cytoplasmic in neonate cells, whereas it is seen more often near the plasma membrane in adult cells. A similar expression pattern was observed when isolated neonatal and adult beta-cells were stained with GLUT2-specific antibodies (Fig. 3C). These results support the idea that one of the reasons why neonatal beta-cells are relatively insensitive to external glucose is that they express fewer GLUT2 transporters in the plasma membrane, with the consequent reduced transmembrane glucose transport.

Ca2+ currents in neonatal and adult beta-cells. Another possible explanation for the deficient insulin release from immature islet beta-cells despite their relatively high insulin content could be an insufficient Ca2+ influx through voltage-gated channels. To evaluate this possibility, Ca2+ currents from individual beta-cells were recorded under the whole cell configuration of the patch-clamp technique, using Ba2+ as charge carrier. Figure 4A shows families of Ba2+ current records obtained from neonatal and adult beta-cells. Figure 4B compares the mean I/V relationships obtained from 12 neonatal and 10 adult cells. In both cases, I/V relationships show two main components of Ba2+ current: a low-voltage-activated component (LVA; with threshold for activation around –40 mV) and a high-voltage-activated component (HVA; with threshold for activation around –20 mV). These two components correspond, respectively, to T-type and L-type VGCCs (3, 15). The main difference between immature and mature beta-cells is that the mean peak current density of the voltage-gated Ba2+ current is ~30% smaller in neonatal beta-cells (10.8 ± 1.0 pA/pF) compared with adult beta-cells (15.5 ± 1.8 pA/pF). As shown in Fig. 4C and Table 1, Ba2+ currents recorded from mature and immature beta-cells showed no significant differences in the parameters of the Boltzmann fit to the voltage dependence of activation.


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  		Table 1. Comparison of Boltzmann parameters calculated for plots of Ba2+ conductances in adult and neonatal beta-cells

 
Expression of mRNAs coding for {alpha}1 subunits of VGCC in neonatal and adult beta-cells. The smaller Ca2+ current density recorded from neonatal beta-cells could result from reduced synthesis of mRNAs coding for the corresponding {alpha}1 subunits. To evaluate this hypothesis, the expression levels of the mRNAs coding for the different Ca2+ channel {alpha}1 subunits present in beta-cells were analyzed by RT-PCR. Interestingly, neonatal beta-cells contain mRNAs coding for all {alpha}1 subunits (Fig. 5). However, the {alpha}1B subunit is considerably more abundant than the other subunits in neonates and also compared with adult cells. Neonatal beta-cells express less mRNA coding for the other subunits compared with adult beta-cells.

Staining pattern of immunoreactivity for insulin and {alpha}1 subunits of VGCC in islets from pancreatic sections. The distribution of insulin-specific immunostaining in intact rat islets was analyzed in frozen sections from neonatal and adult pancreatic tissue. The same sections were also immunostained with specific antibodies raised against different {alpha}1 subunits of VGCC (see RESEARCH DESIGN AND METHODS). Islets can be easily distinguished from acinar tissue in confocal images of frozen sections. In general, and regardless of the developmental stage, insulin-specific fluorescence is more abundant in the central part of the islet (see Figs. 6 and 7, left columns).

Considerable cell-to-cell differences in the intensity of insulin-specific immunostaining are noticeable, especially in adult islets. This heterogeneity is less conspicuous in neonatal islets. In general, beta-cells are immunoreactive to all {alpha}1 subunits examined, both in the neonatal and in adult tissue (Figs. 6 and 7, middle columns), but in contrast to insulin staining, {alpha}1-specific immunoreactivity appears more evenly distributed throughout the islet. When confocal images of insulin and {alpha}1 fluorescence are compared, the islet footprint in the {alpha}1 (green) image appears bigger than the insulin (red) image (Fig. 6: {alpha}1C and {alpha}1D, adult; Fig. 7: {alpha}1B adult). When red and green images are merged (Figs. 6 and 7, right columns), it is clear that {alpha}1 staining is found throughout the islet, whereas insulin staining is typically located in the core. These results show that {alpha}1 subunits of VGCC are expressed both in insulin-positive beta-cells and in the insulin-negative {alpha}- and {delta}-cells of the islet.

Subcellular distribution of immunoreactivity of {alpha}1 subunits of VGCC in cultured beta-cells. Despite the fact that neonatal cells display smaller Ca2+ currents and have fewer copies of {alpha}1C, {alpha}1D, and {alpha}1G mRNA subunits than adult cells, a comparison of {alpha}1 immunoreactivity between neonatal and adult islets showed differences. Quantification of single-cell immunoreactivity from neonatal and adult rats failed to reveal differences in the expression of {alpha}1 subunits (data not shown). We then explored the subcellular localization of immunoreactivity in single cultured cells.

Figure 8 shows examples of digital fluorescence micrographs obtained from isolated cells immunostained for {alpha}1D subunits of VGCC. It is clear that the immunoreactivity to {alpha}1 is more intense in the cell periphery than in the cytoplasm. This annular fluorescence pattern, perhaps reflecting the predominant distribution of {alpha}1 subunits in the plasma membrane, was analyzed in 25 cells per condition (see RESEARCH DESIGN AND METHODS), and the corresponding M/C ratio was computed. Table 2 shows that, in neonatal cells, {alpha}1B, {alpha}1C, and {alpha}1D are expressed almost equally in both membrane and cytoplasm compartments, whereas {alpha}1G is expressed mostly in the plasma membrane. In contrast, {alpha}1C, {alpha}1D, and {alpha}1G are preferentially localized in the plasma membrane compartment in adult beta-cells. These findings support the notion that Ca2+ currents recorded in adult beta-cells are larger than in neonatal beta-cells, because the corresponding {alpha}1 subunits of VGCC tend to locate more in the plasma membrane and less in the cytoplasm.


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  		Table 2. Subcellular distribution of {alpha}1 subunits of VGCC in cultured beta-cells

 

   DISCUSSION
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 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Glucose homeostasis during fetal development in mammals is mainly achieved by the mother, because fetal pancreatic beta-cells barely respond to high glucose concentrations by increasing insulin secretion (21, 26). In this stage, insulin secretion is scarce and monophasic, similar to the pattern that is observed in the onset of type 2 diabetes (9, 48, 49). After birth, changes in feeding and nutritional metabolism entail a fast adaptation of the organism to the new situation. To our knowledge, the results of the present work comprise the first comparison between functionally immature neonatal beta-cells and adult cells, giving special attention to the factors that contribute to immaturity of insulin secretion.

Glucose-induced insulin secretion by adult pancreatic beta-cells is a biphasic process that depends on the rise of [Ca2+]i. The membrane potential of beta-cells in a basal glucose concentration (5.6 mM) oscillates near the resting level (35). When extracellular glucose rises (above 6.5 mM), it enters the cells through GLUT2-type transporters. Then glucose is metabolized and the ATP/ADP ratio increases, leading to the closure of ATP-sensitive K+ channels and a slow membrane depolarization. This process activates Na+ and Ca+2 channels, which leads to a fast membrane depolarization and a plateau level, on top of which fire action potentials, and finally the membrane repolarizes. The increase in [Ca2+]i promotes the exocytosis of insulin granules (34, 37, 38).

The present study demonstrates that neonatal rat beta-cells do not discriminate between different glucose concentrations and secrete less insulin than adults. This deficiencies result, at least in part, from the following.

Neonate beta-cells express less insulin mRNA than adults, suggesting reduced insulin biosynthesis. Moreover, as neonate cells secrete less insulin, even when glucose concentration is high, they are more immunoreactive to the hormone than adults, suggesting that they tend to accumulate the hormone.

Neonatal beta-cells express all Ca2+ channels subtypes that are observed in the adults. However, neonatal Ba2+ current density is lower and levels of mRNA coding {alpha}1C, -D, and -G subunits of Ca2+ channels are reduced compared with adult beta-cells. Additionally, the percentage of L-type channels, which have been described as the most important for insulin secretion, is reduced in neonate beta-cells. These observations may explain why insulin secretion in neonate beta-cells is weaker than in adults.

In addition, neonate beta-cells express fewer GLUT2 transporters in the membrane than adult cells. This observation suggests that maximum glucose transport could be reduced in neonates and become saturated at high glucose concentrations. It is interesting to observe that the low expression of GLUT2 in the membrane of immature beta-cells reflects a physiological state, whereas a reduced expression of GLUT2 transporters can also be observed in type 2 diabetes (9). In both cases, the deficiency results in the inability to discriminate between different glucose concentrations.

Neurons and myocytes exhibit a changing pattern of Ca2+ channel expression during development (19, 24, 31, 45). This type of regulation has not been previously reported in pancreatic beta-cells, and it is interesting because it could be related to the functional maturation of beta-cells.

It has been reported that L-type calcium channels tend to be located near insulin granules in beta-cells (5). Since glucose-induced insulin secretion follows a biphasic time course, it has been suggested that this reflects the sequential release of distinct pools of granules, which are associated with different subtypes of L-type calcium channels (1, 2).

Although mRNAs for {alpha}1C (Cav1.2) and in particular {alpha}1D (Cav1.3) and their respective channel proteins have been found in all tested insulin-secreting cells and islets, their distinct contribution to insulin exocytosis has not been thoroughly studied in different species and remains controversial (51). For example, it has been reported that, in mouse beta-cells, the {alpha}1C subunit is necessary for the first phase of secretion (35), whereas the {alpha}1D subunit participates mainly in response to 3 mM glucose (41). Moreover, the latter could be important for postnatal beta-cell generation (27). In INS-1 cells, LD-type Ca2+ channels are preferentially coupled to glucose-stimulated insulin secretion (23).

In this study, we observed a lower expression of both mRNAs for {alpha}1D and {alpha}1C subunits in neonate rat beta-cells compared with adult; moreover, the levels of expression these channel types in the membrane are less abundant in neonates than in adults. These observations could partially explain why neonate beta-cells, even though their hormone content is higher, secrete less insulin than adult beta-cells. We (8, 36) have previously observed that in adult beta-cells nifedipine completely blocked glucose-stimulated insulin secretion without affecting basal glucose secretion. In this study, we measured the effect of different Ca2+ channel blockers in immature beta-cells without success (data not shown). This lack of effect could be due to their insensitivity to high glucose concentrations.

Insulin-secreting cell lines express functional N-type Ca2+ channels, which are important for insulin secretion (17, 43); however, it has not been clearly established whether primary pancreatic beta-cells express this type of channel, and their role in the glucose-induced insulin secretion is still under debate (12, 22, 34, 43, 45). We observed expression of {alpha}1B mRNA and their proteins in neonatal and adult beta-cells. However, only about one-half of them are located in the plasma membrane at both stages. This observation could explain the contradictory results reported in the literature. It is possible that the expression of these channels in the membrane can be modulated by different conditions yet to be investigated. Finally, we found that the expression of mRNA {alpha}1G subunit is slightly less in neonatal beta-cells compared with adults, whereas nearly 64% of T-type Ca2+ channels are located in the plasma membrane at both ages. In INS-1 cells, the activity of T-type Ca2+ channels enhances excitability of cells, as has also been observed in other cell types (29), and facilitates insulin secretion (4).

Our observations suggest that diabetic cells behave like immature beta-cells in several regards; however, more experiments are necessary to confirm this hypothesis. Characterizing neonatal beta-cell physiology and unrevealing how they mature can contribute toward a better understanding of the physiopathology of the type 2 diabetes where a low expression of Ca2+ channels and GLUT2 transporters and deficient insulin secretion have been reported.


   GRANTS
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by grants from Dirección General de Asuntos del Personal Académico de la Universidad Nacional Autónoma de México (DGAPA) 203903 and Consejo Nacional de Ciencia y Tecnologia (CONACYT) D39822Q to M. Hiriart, and DGAPA IN226403 and CONACYT 42662-Q to A. Hernández-Cruz. V. Navarro-Tableros is a CONACYT fellow. T. Fiordelisio received support from the Programa de Retención y Repatriación 2006, CONACYT.


   ACKNOWLEDGMENTS
 
We are grateful to Carmen Sánchez-Soto, Félix Sierra, and Federico Jandete-García for technical assistance and to Claudia Rivera-Cerecedo for providing animal management and care. We are also grateful to the Unit of Molecular Biology and Unit of Microscopy and to Ana Escalante and Francisco Pérez for computing assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. Hiriart, Depto de Biofísica, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Ciudad Universitaria, AP 70-253, Coyoacán, México, DF 04510, México (e-mail: mhiriart{at}ifc.unam.mx)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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M. Hiriart and L. Aguilar-Bryan
Channel regulation of glucose sensing in the pancreatic {beta}-cell
Am J Physiol Endocrinol Metab, December 1, 2008; 295(6): E1298 - E1306.
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